Crystals of membrane proteins

ABSTRACT

A polypeptide in crystalline form comprises a G-protein coupled receptor (GPCR) with an IC3 loop substituted by an amino acid residue sequence of lysozyme.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/739,134 filed on Sep. 23, 2010 which is a U.S. National Stage of PCT/US2008/080847, filed Oct. 22, 2008, and claims benefit of U.S. provisional application 60/999,951, filed Oct. 22, 2007; U.S. provisional application 61/000,325, filed Oct. 24, 2007; U.S. provisional application 61/060,107, filed Jun. 9, 2008; and U.S. provisional application 61/194,961, filed Oct. 1, 2008, each of which is incorporated herein by reference, in its entirety, for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. GM73197 awarded by the National Institutes of Health; GM74691 and GM62411 awarded by the Protein Structure Initiative; Y1-CO-1020 awarded by the National Cancer Institute; and Y1-GM-1104 awarded by the National Institute of General Medical Sciences. The government has certain rights in this invention.

Coordinates and structure factors have been deposited in the Protein Data Bank with identification code 2RH1.

BACKGROUND OF THE INVENTION

Naturally occurring polypeptides or proteins often fold into complex, three-dimensional shapes that determine both chemical and physiological functionality. Thus a thorough understanding of proteins necessarily involves a detailed representation of their spatial topography. The field of protein crystallography has flourished over the last 20 years resulting in a rapid increase in the knowledge bas of protein structure enabling great strides in other disciplines including biochemistry, pharmaceutical development and cell biology. However, the structural biology field has largely been restrained to working with protein that is naturally soluble in aqueous media, or made soluble by incorporation into surfactant micelles. The present invention provides methods and compositions that allow for the study of membrane-embedded proteins (i.e., integral membrane proteins) in a more natural membrane bilayer environment. The present invention enable a more detailed analysis of important classes of membrane-embedded polypeptides that play key roles in a variety of cellular processes including energy and signal transduction.

SUMMARY OF THE INVENTION

Other objects, features and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference for all purposes and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

In one aspect the invention provides compositions, e.g., useful for the crystallization of membrane protein. In some embodiments, the composition is suitable for lipidic cubic phase crystallization. In some embodiments the compositions contain 10-60% v/v of a polyethylene glycol, 0.01-0.5 M of a salt, 1-20% v/v of an organic compound, and 1-50% w/w of a lipid additive in a host lipid. In some embodiments of the compositions, the protein to be crystallized is present at a concentration of 1 to 100 mg/mL. In some embodiments, the protein to be crystallized is present at a concentration of 50 mg/mL.

In some embodiments of the compositions of the inventions, the polyethylene glycol is PEG or modified PEG at a molecular size of 10-8000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-8000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-2000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400-1000. In some embodiments, the PEG or modified PEG has an average molecular weight of 400. Examples of modified PEG include but are not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenated castor oil, PEG castor oil, PEGcorn oil, PEG caprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester and methoxypoly(ethylene glycol) (mPEG).

In some embodiments of the compositions, the salt is a sulfate salt. In some embodiments, the sulfate salt is sodium sulfate. In some embodiments, the salt is present at a concentration of 0.1-0.5 M. In some embodiments, the salt is present at a concentration of 0.1-0.2 M. In some embodiments, the compositions of the invention contain a buffer. In some embodiments, the buffer is present at a concentration of 0.01-0.5 M. In some embodiments, the buffer is present at a concentration of 0.1-0.2 M. In some embodiments, the buffer is present at a concentration of 0.1 M. In some embodiments, the buffer is Bis-tris propane. In some embodiments, the buffer has a pH 6.5-7.0. In some embodiments of the compositions, an organic compound is present at a concentration of 1-10% v/v. In some embodiments, the organic compound is present at a concentration of 5-7% v/v. In some embodiments, the organic compound is 1,4-butanediol.

In some embodiments of the compositions, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of lipid additives include but are not limited to cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments, the host lipid is monoolein.

In some embodiments of this aspect, the composition of the invention comprises 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid.

In another aspect the invention includes compositions suitable for lipidic cubic phase crystallization. In some embodiments, the compositions of the inventions contain a lipid additive. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.

In another aspect the invention includes methods for crystallization of membrane proteins. In some embodiments, the method for crystallization of membrane proteins comprises adding a lipid additive to a lipidic cubic phase. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.

In another aspect, the invention provides for methods of crystallization of a protein. In some embodiments of this aspect, the method comprises, providing said protein in a lipidic cubic phase composition, filling a plate comprising a material that does not interfere with imaging (such as, e.g., a transparent glass or plastic) with said composition, placing said plate containing said composition under conditions suitable for crystallization of said protein and detecting the presence of a crystal of said protein in said plate. In some embodiments, the method further comprises covering said plate with a second plate comprising a material that does not interfere with imaging (such as, e.g., a transparent glass or plastic).

In some embodiments of the methods, the protein is a non-colored protein. In some embodiments, the protein is a G protein-coupled receptor (GPCR). In some embodiments, the protein comprises a β₂AR, a CXCR4, or a human adenosine A_(2A) receptor. In some embodiments the protein comprises a stabilizing point mutation or a T4 lysozyme fusion or both.

In some embodiments of the methods, the lipidic cubic phase composition comprises a lipid additive. Examples of lipid additives include, but are not limited to, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin. In some embodiments, the lipid additive is cholesterol. In some embodiments, the lipid additive is present at a concentration of 1-50% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 1-20% w/w in a host lipid. In some embodiments, the lipid additive is present at a concentration of 8-10% w/w in a host lipid. Examples of host lipids include, but are not limited to monopalmitolein, monovaccenin and monoolein. In some embodiments the host lipid is monoolein.

In some embodiments of the methods, the first plate and second plate are made of glass. In some embodiments, the plate allows for the control of crystallization conditions, such as the humidity and temperature of said lipidic cubic phase composition.

In some embodiments of the methods, the crystals are harvested directly from the plate. In some embodiments of the methods, the crystals are harvested between the cubic and the sponge phase of the lipidic cubic phase composition. In some embodiments of the methods, the crystals are harvested directly from said lipidic cubic phase composition and placing said crystals in liquid nitrogen.

In another aspect the invention provides methods for screening a crystal present in a liquid cubic phase composition. In some embodiments, the method comprises exposing the composition to a first beam and determining a change of the first beam, exposing the composition to a second beam and determining a change of the second beam, and identifying an area where the crystal is present in said composition. Examples of changes in the beams, include but are not limited to, change in direction and/or intensity of the beams. In some embodiments the crystals are non-colored.

In some embodiments of the methods, the first beam and second beam are attenuated. In some embodiments the beams are attenuated 10 times. In some embodiments, the first beam is a slitted 100×25 μm beam. In some embodiments, the methods comprise exposing said lipidic cubic phase composition to a third beam. In some embodiments, the methods comprise exposing said lipidic cubic phase composition to up to ten extra beams. In some embodiments, the exposure of composition to the beams is 2 seconds. In some embodiments, the beams are beams of visible light.

In another aspect the invention includes a crystal of a membrane protein. In some embodiments, the invention includes a crystal of a G protein-coupled receptor (i.e., a “GPCR”) non-covalently bound to a ligand. In some embodiments, the extracellular domain of said crystalline GPCR is resolvable by X-Ray crystallography. In some embodiments, the ligand is a diffusible ligand.

In some embodiments of this aspect, the volume of the crystal is greater than 15×5×1 μm. In some embodiments, the volume of said crystal is greater than 30×5×5 μm. In some embodiments of this aspect, the volume of crystal is greater than 40×20×5 μm. In some embodiments, volume of the crystal is estimated assuming that each of the stated dimensions are orthogonal so that the volume estimate is the product of the dimensions. In some embodiments, the crystal is suitable for X-ray crystallography. In some embodiments, X-ray crystallographic analysis can be carried out to determine the structure of a protein comprising said crystal.

In some embodiments of this aspect, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å. In some embodiments, the crystal diffract to a resolution of 2.0 to 5.0 Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.4 Å.

In some embodiments, the G protein-coupled receptor is a β₂AR protein, a CXCR4 protein, or a human adenosine A_(2A) receptor protein.

In another aspect the invention provides for a crystal of β₂AR. In some embodiments, the structure of an extracellular domain of said crystal is capable of being resolved by X-ray crystallography. In some embodiments, the crystal comprises 442 amino acids, a palmitic acid covalently bound to Cys341 and an acetamide molecule bound to Cys265^(6.27), a diffusible ligand, up to 10 molecules a lipid additive, up to five salt ions and up to 10 butanediol molecules. In some embodiments, the lipid additive is cholesterol. In some embodiments, the crystal comprises three cholesterol molecules. In some embodiments, the salt ion is a sulfate ion. In some embodiments, the crystal comprises two sulfate ions. In some embodiments, the diffusible ligand is carazol. In some embodiments the crystal comprises two butanediol molecules. In some embodiments of this aspect, the volume of the crystal is greater than 15×5×1 μm. In some embodiments, the volume of said crystal is greater than 30×5×5 μm. In some embodiments of this aspect, the volume of the crystal is greater than 40×20×5 μm. In some embodiments the volume of the crystal is estimated by assuming that each dimension is orthogonal to the other dimensions so that the volume is the product of the three lengths. In some embodiments, the crystal is suitable for X-ray crystallography. In some embodiments, the structure of a β₂AR protein can be determined from said crystal using X⁻-ray crystallographic analysis. In some embodiments, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments of this aspect, the crystal is crystallized using liquid cubic phase crystallization. In some embodiments, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In some embodiments, the crystal diffract to a resolution of 1.0 to 10.0 Å. In some embodiments, the crystals diffract to a resolution of 2.0 to 5.0 Å. In some embodiments, the crystal diffract to a resolution of 2.2 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 3.2, 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.8, 2.6 or 2.4 Å. In some embodiments, the structure of said crystal is solved and refined at resolution of less than about 2.4 Å.

In another embodiment, the invention provides a composition for lipidic cubic phase crystallization of a membrane protein, comprising a polyethylene glycol or modified polyethylene glycol; 0.01-1M of a salt; a host lipid; a lipid additive, wherein said lipid additive is present at 10-60% v/v ratio relative to the host lipid; a buffer; and 1 to 100 mg/ml of a membrane protein. In a related embodiment, the polyethylene glycol is PEG or modified PEG, wherein said PEG or modified PEG has an average molecular weight of 200-20,000, 400-8000, or 400-2000. In yet another related embodiment, the PEG or modified PEG in the composition has an average molecular weight of 400. In another related embodiment, the salt is selected from the group consisting of a sulfate salt, a citrate salt, a malonate salt, a tartrate salt, an acetate salt, and a formate salt. In certain embodiments of the composition, the salt is present at a concentration of 0.1-0.2 M. In another related embodiment, the buffer is present at a concentration of 0.05-0.5 M in the composition. In certain embodiments, the buffer is Bis-tris propane or sodium citrate. In other related embodiments of the composition, the buffer has a pH between 4.5-8.0.

In still other related embodiments of the composition for lipidic cubic phase crystallization of a membrane protein, the composition further comprising an alcohol present at a concentration of 1-10% v/v or 5-7% v/v. In certain embodiments, the alcohol is a diol or triol. In other embodiments, the alcohol is 1,4-butanediol or 2,6-hexanediol.

In still other related embodiments of the composition for lipidic cubic phase crystallization of a membrane protein, the lipid additive is present at a concentration of 1-20% w/w in a host lipid or 8-10% w/w in a host lipid. In yet another related embodiment of the composition, the lipid additive is selected from the group consisting of 2-monoolein, phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid, dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoyl phosphatidylcholine (“DOPC”), Asolectin, and a sterol. In still other embodiments, the lipid additive is a sterol. In related embodiments, the lipid additive is cholesterol.

In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein, the host lipid is selected from the group consisting of monopalmitolein, monovaccenin and monoolein. In a related embodiment, the host lipid is monoolein. In still another related embodiment, the membrane protein to be crystallized in said composition is present at a concentration of 1 to 100 mg/mL. In yet another embodiment, the membrane protein to be crystallized in said composition is present at a concentration of 40-60 mg/mL.

In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein membrane protein is a G-protein coupled receptor, such as a β₂AR protein, a human adenosine A2A receptor protein, a CXCR4-T4L protein, or a β₂AR-T4L protein. In related embodiments, the β₂AR^((E122W)), a β₂AR^((E122W))-T4L, a human G-protein coupled receptor is a protein comprising or consisting of a adenosine A2A receptor-T4L, a CXCR4-T4L or β₂AR-T4L. In still another related embodiment of the composition for lipidic cubic phase crystallization of a membrane protein, the composition comprises a ligand selected from the group consisting of carazolol, timolol, alprenolol, and clenbutorol.

In another embodiment, the invention provides a method of generating crystals of a membrane protein comprising: mixing a lipid additive with a host lipid to form a lipid mixture, wherein said lipid additive is selected from the group consisting of a sterol, DOPE, DOPE-Me, DOPC, and Asolectin, and wherein said lipid additive is 5 to 20% w/w in said host lipid; and combining said lipid mixture with a membrane protein solution under conditions suitable for the formation of a lipidic cubic phase composition. In a related embodiment of the method, said protein is a non-colored protein. In a related embodiment, the amount of said lipid additive is 8 to 10% w/w in said lipid. In another related embodiment, the invention further comprises: filling a plate with said lipidic cubic phase composition, wherein said plate is compatible with imaging; placing said plate containing said lipic cubic phase composition under conditions suitable for crystallization of said protein; and detecting the presence of a crystal of said protein in said plate. In another embodiment, the method further comprises covering said plate with a second plate.

In a related embodiment of the method of generating crystals of a membrane protein, the protein is a GPCR. In yet another related embodiments, the protein comprises aβ₂AR. In yet another related embodiment, the β₂AR protein is selected from the group consisting of β₂AR^((E122W)), β₂AR^((E122W))-T4L, and β₂AR-T4L. In yet another related embodiment, the GPCR is a human adenosine A2A receptor or a CXCR4 receptor where the proteins may comprise, in still other related embodiments, a T4 lysozome.

In yet another related embodiment of method of generating crystals of a membrane protein, the lipid additive is present at a concentration of 1-20% w/w or 8-10% w/w in a host lipid. In yet another related embodiment of the method, the second plate comprises a glass. In yet another related embodiment, the method further comprises harvesting crystals directly from said plate. Another related embodiment of the method comprises harvesting crystals from between the cubic and the sponge phase of said lipidic cubic phase composition. In another related embodiment, the method comprises harvesting crystals directly from said lipidic cubic phase composition and placing said crystals in liquid nitrogen. In yet another related embodiment, the method comprising a step of soaking into said crystal a diffusable ligand or candidate ligand.

The invention also provides a method of screening a crystal of a GPCR present in a liquid cubic phase composition comprising: preparing a liquid cubic phase composition comprising a GPCR protein, a host lipid, and a lipid additive; exposing said composition to a first X-ray beam and determining a change in direction or intensity of said first X-ray beam; exposing said composition to a second beam and determining a change in direction or intensity of said second X-ray beam; identifying an area where said GPCR crystal is present in said composition; and exposing said identified area to at least a third X-ray beam. In a related embodiment, the first beam is a slitted 100×25 μm beam. In another related embodiment, the crystal is colorless. In yet another embodiment, the GPCR crystal is β₂AR^((E122W))-T4L, β₂AR^((E122W)), β₂AR, AK or β₂AR-T4L protein. In related embodiments, the crystal is human adenosine A2A receptor or a CXCR4 receptor where the proteins may comprise, in still other related embodiments, a T4 lysozome.

In another embodiment, the invention provides a crystal of a human β₂AR protein wherein the extracellular loop ECL2 of said β₂AR is sufficiently ordered to produce interpretable electron density in a crystallographically-derived electron density map. In yet another related embodiment, each β₂AR molecule in said crystal comprises three non-covalently bound cholesterol molecules and at least one salt ion. In yet another related embodiment, the at least one salt ion is a sulfate ion. In yet another related embodiment, each β₂AR molecule in said crystal said crystal comprises two sulfate ions. In yet another related embodiment, each β₂AR molecule in said crystal said crystal further comprises carazol. In yet another related embodiment of the crystal, each β₂AR molecule in said crystal comprises between 1 and 10 butanediol molecules. In yet another related embodiment of the crystal, the volume of said crystal exceeds 15×5×1 μm, 30×5×5 μm or 40×20×5 μm. In yet another related embodiment, the specific surface area of the crystal is 0.0001-5 m²/g. In yet another related embodiment, the crystal is crystallized using liquid cubic phase crystallization. In yet another related embodiment, the crystal is obtainable by harvesting the crystal from a glass sandwich plate. In yet another related embodiment, the crystal diffracts to a resolution of 2.0 to 10.0 Å, 2.0 to 5.0 Å, or 2.2 to 2.4 Å. In yet another related embodiment, the structure of said crystal is solved and refined at a resolution higher than about 3.2, higher than about 2.8, or higher than about 2.4 Å.

In yet another embodiment, the invention provides a crystalline form of a human β₂AR protein having an atomic arrangement of coordinates comprising the β₂AR coordinates set forth in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance).

In another embodiment, the invention provides a crystalline form of a human β₂AR protein, where said form has unit cell dimensions of a=106.3 Angstroms, b=169.2 Angstroms, and c=40.2 Angstroms. In a related embodiment, said β₂AR protein is β₂AR-T4L. In another related embodiment, the β₂AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of a human β₂AR protein, wherein said space group of said crystalline form is C2. In a related embodiment, said β₂AR protein is β₂AR-T4L. In another related embodiment, the β₂AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of a human β₂AR protein, wherein said crystalline form diffracts X-rays to a resolution of 2.4 Angstroms. In a related embodiment, said β₂AR protein comprises a point mutation that stabilizes said β₂AR protein. In another related embodiment, said β₂AR protein is β₂AR-T4L. In a related embodiment, the β₂AR-T4L crystal further comprises a carazolol ligand.

In another embodiment, the invention provides a crystalline form of a human β₂AR protein wherein each β₂AR molecule in said crystal comprises 442 amino acids, a palmitic acid covalently bound to Cys341, an acetamide molecule bound to Cys265^(6.27), a diffusible ligand, one to ten molecules of a lipid additive, one to five salt ions and one to ten butanediol molecules.

In another embodiment, the invention provides a method of identifying a compound that binds to a ligand binding site of a human β₂AR protein, comprising comparing a set of three-dimensional structures representing a set of candidate compounds with a three-dimensional molecular model of said ligand binding site, comprising: receiving a three-dimensional model of a ligand binding site on said human β₂AR protein, wherein said three-dimensional model of said ligand binding site comprises atomic co-ordinates for a plurality of ligand-binding residues, wherein said atomic co-ordinates are taken from Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance); determining, for each of the set of compound three-dimensional models, a plurality of distance values indicating distances between said atomic co-ordinates of said candidate compound of the set of candidate compounds and said atomic coordinates of said ligand-binding residues comprising said ligand binding site; determining, for each of the set of compound three-dimensional models, a binding strength value based on the plurality of distance values determined for the compound three-dimensional model, wherein the binding strength value indicates the stability of a complex formed by said human β₂AR protein and a compound represented by the compound three-dimensional model; and storing a set of results indicating whether each candidate compound binds to the three-dimensional model based on the binding strength values. In a related embodiment, said ligand-binding residues comprise a plurality of residues selected from the group consisting of Y199, A200, S204, T118, V117, W286, Y316, F290, F289, N293, W109, F193, and Y308. In another related embodiment, said ligand-binding residues comprise a plurality of residues selected from the group consisting of W109, V117, T118, F193, Y199, A200, W286, F289, F290, Y316. In another related embodiment of the method of identifying a compound that binds to a ligand binding site of a human β₂AR protein, said binding strength value is based on one or more of a hydrogen bonding strength, a hydrophobic interaction strength, or a Coulombic interaction binding strength. In another related embodiment, one or more of said receiving, determining, or storing steps is carried out using a commercially-available software program. In yet another related embodiment, the commercially-available software program is selected from the group consisting of DOCK, QUANTA, Sybyl, CHARMM, AMBER, GRID, MCSS, AUTODOCK, CERIUS II, Flexx, CAVEAT, MACCS-3D, HOOK, LUDI, LEGEND, LeapFrog, Gaussian 92, QUANTA/CHARMM, Insight II/Discover, and ICM. In yet another related embodiment, the method further comprising the step of contacting a human β₂AR protein with a molecule comprising an identified candidate compound. In yet another related embodiment, the molecule further comprises a moiety capable of competitively displacing a ligand from said human β₂AR protein, wherein said ligand binds to said ligand binding site of said human β₂AR protein. In yet another related embodiment, the method further comprising characterizing a binding interaction between said human β₂AR protein and said molecule comprising said identified candidate compound, and storing a result of said characterizing. In yet another related embodiment, said characterization comprises determining an activation of a function of said human β₂AR protein, an inhibition of a function of said human β₂AR protein, an increase in expression of said human β₂AR protein, a decrease in expression of said human β₂AR protein, a displacement of a ligand bound to said ligand binding site, or a stability measure for said human β₂AR protein.

In another embodiment, the invention provides a method for selecting a library of potential modulators of β₂AR to be screened, comprising calculating a structure of a first potential modulator using at least a portion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance), correlating said structure of said first potential modulator with a library of modulators identified as comprising said structure said first potential modulator, and storing or transmitting information about the identified library.

In yet another embodiment, the invention provides a method of solving the structure of a crystalline form of a protein, comprising: using at least a portion of the structure co-ordinates of Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) to solve the structure of the crystalline form of a test protein, wherein said test protein has significant amino acid sequence homology to any functional domain of β₂AR; and transmitting or storing data descriptive of the structure of said test protein.

In another embodiment, the invention provides a method of identifying from a set of candidate compound three-dimensional models a compound that binds to a ligand binding site of a GPCR or β₂AR protein comprising: receiving a three-dimensional model of a ligand binding site on said GPCR or β₂AR protein, wherein said three-dimensional model of said ligand binding site comprises atomic co-ordinates for a plurality of ligand binding residues; determining, for each candidate compound of the set of candidate compound three-dimensional models, a plurality of distance and angle values indicating distances and angles between atomic co-ordinates of said candidate compound of the set of candidate compound three-dimensional models and said ligand binding site comprising atomic coordinates of said ligand-binding residues; determining, for each of the set of candidate compound three-dimensional models, a binding strength value based on the plurality of distance and angle values determined for the candidate compound three-dimensional model, wherein the binding strength value indicates the stability of a complex formed by said human GPCR or β₂AR protein and a compound represented by the compound three-dimensional model; storing a set of results indicating whether each candidate compound binds to the three-dimensional model based on the binding strength values; searching a database of small organic molecules for compounds exhibiting shape, chemistry, or electrostatic similarity with the candidate compounds indicated to bind to the three-dimensional model based on the binding strength values; and identifying the set of small organic molecules exhibiting shape, chemistry, or electrostatic similarity with the candidate compounds indicated to bind to the three-dimensional model based on binding strength values as likely to also bind to the GPCR or β₂AR the database of small organic molecules is the available chemicals database. In a related embodiment, the shape, chemistry or electrostatic similarity is determined using a program selected from the group consisting of BROOD (openeye), EON (openeye), ROCS (openeye), ISIS Base, and SciFinder.

In another embodiment, the invention provides a method of identifying a ligand that binds to a membrane protein comprising: preparing a lipid meso phase, wherein said lipid meso phase composition comprises (1) a host lipid; (2) said membrane protein; (3) a lipid additive selected from the group consisting of consisting of a sterol, cholesterol, DOPE, DOPE-Me, DOPC, and Asolectin, wherein said lipid additive is 1 to 50% w/w in a lipid host; subjecting said lipid meso phase to humidity and temperature conditions to grow crystals of said membrane protein; contacting said membrane protein crystals with diffusible ligands or a mixture of diffusible ligands; determining the three-dimensional structure of said diffusible ligand contacted membrane protein crystals by X-ray crystallography to obtain an electron density map; and identifying bound ligands by inspection of the electron density map. In a related embodiment, the ligands are substantially insoluble in water.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1. Crystals of β₂AR-T4L obtained in 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid. FIG. 1 a shows β₂AR-T4L crystals in the crystallization mixture drop (upper left) and in the loop. FIG. 1 b shows crystals in the “sponge” phase.

FIG. 2. Before (top) and after (bottom) images of a lipidic cubic phase crystal harvested directly from a well in a previously sealed glass sandwich plate, according to the method described herein (see, e.g., Example 1).

FIG. 3. Diffraction pattern (2.8 Å resolution) from β₂AR-T4L crystals grown in lipid cubic phase. The crystal size was approximately 25×5×5 Å; space group C2 (a=106.8 Å, b-169.5 Å, c=40.5 Å; (i=105.3°, α=γ=90°. Beam diameter was 10 μm, exposure 10 s, oscillation: 1°.

FIG. 4. Gallery of crystals of various GPCRs obtained using LCP/cholesterol mixtures and in combination with a variety of ligands. The top panel corresponds to non-optimized initial hits, whereas the bottom panel shows diffraction quality for optimized crystals. From left to right, β₂AR-T4L (bound to carazolol), diffracted to 2.4 Å resolution; β₂AR(E122W)T4L (bound to carazolol), diffracted to 3.5 Å resolution; β₂AR(E122W)T4L (bound to alprenolol), diffracted to 3.5 Å resolution; β₂AR(E122W)T4L (bound to timolol), diffracted to 2.8 Å resolution; β₂AR(E122W) (bound to carazolol); β₂AR(E122W)T4L (bound to clenbuterol), diffracted to 6 Å, anisotropic; human β_(2A) adenosine receptor-T4L (bound to ZM241385), diffracted to 2.6 Å resolution.

FIG. 5. A. β₂AR-T4L Crystals grown from bicelle conditions. B. Diffraction image from bicelle grown microcrystals of β₂AR-T4L recorded using 10 μm minibeam on 231D-B beamline at APS. Black circle is drawn at resolution 3.5 Å.

FIG. 6. A. Microcrystals of β₂AR-T4L grown in lipidic mesophase. B. Diffraction image from lipidic cubic phase grown microcrystals of β₂AR-T4L recorded using a 10 μm minibeam on 231D-B beamline at APS. The white circle is drawn at resolution 2.2 Å.

FIG. 7. Detailed representation of the carazolol binding site in β₂AR-T4L. FIGS. 7A, B, and C are representations of the electron density of the ligand binding site at three different orientations. Residues are labeled by their Ballesteros-Weinstein numbers as superscripts. Electron density is contoured at 1σ from a 2 F_(o)-F_(c) difference map. Both B and C are generated by rotating the field of view 90° about the y-axis clockwise and counterclockwise respectively.

FIG. 8. Electron density of: A. Cholesterol molecules shown with a F_(o)-F_(c) electron density contoured at 2σ omitting the lipid from phase calculation, palmitic acid is also shown. B. Helix-kinked region with 2F_(o)-F_(c) electron density contoured at 1.5σ.

FIG. 9. Overall fold of the β₂AR-T4L fusion with its predicted orientation in the plasma membrane and key intramolecular interactions. A. Stereoview of the overall fold of β₂AR-T4L. The receptor and T4L are colored gray and green, respectively. Carazolol is colored blue and the lipid molecules bound to the receptor are colored yellow. B. The receptor is aligned to a rhodopsin model that was positioned in a lipid membrane (boundaries indicated by horizontal black lines) as found in the orientations of proteins in membranes (OPM) database (M. A. Lomize et al., Bioinformatics 22, 623 (2006)). T4L is fused internally into the third intracellular loop of β₂AR and maintains minimal intramolecular packing interactions by tilting away from the receptor. C. Specific intramolecular interactions between β₂AR and T4L are represented.

FIG. 10. Crystal packing interactions in the lipidic mesophase crystallized β₂AR-T4L. A. There are four main contact areas, two of which are mediated by T4L in the plane of the membrane with itself through a two-fold symmetry axis and translation. The third interaction is normal to the membrane plane between T4L and lumen exposed loops of β₂AR. The fourth interaction is generated by the two-fold symmetry axis, packing one receptor to receptor in the plane of the membrane. B. The receptor crystal packing interface is composed mainly of lipids with two cholesterol molecules and two palmitic acid molecules forming the majority of the interactions. A network of ionic charge interactions exists on the cytoplasmic end of the interface forming the only inter-receptor protein contacts. C. Comparison between β₂AR-T4L and rhodopsin (PDB ID Code 2135) parallel receptor association interface. Helices I (blue) and VIII (magenta) are highlighted in both structures. Only one monomer is shown for each receptor representation along with helices I′ and VHF only from the opposing symmetry related molecule. The rhodopsin interface is twisted significantly relative to β₂AR-T4L resulting in a significant offset from the parallel orientation required for a physiological dimer interface. β₂AR-T4L associated monomers are in a highly parallel orientation.

FIG. 11. Surface representation of β₂AR colored by calculated charge from red (−10 k_(b)T/e_(c)) to blue (+10 k_(b)T/e_(c)) using a dielectric constant of 70. A. Three main areas of interest are indicated. The binding site cleft is negatively charged as is a groove between helices III, IV and V. The third region is an overall positive charge in the region of the ionic lock and DRY motif on the cytoplasmic face. The overall result is a highly polarized molecule that may utilize its negative charge to facilitate binding of catecholamine ligands. The presence of a negative charge in the groove between helices III, IV and V is unexpected as it is in the middle of the lipid membrane. This charge may be partially derived from the presence of an unpaired glutamate at position 122^(3.41). The effective charge in this region is likely greater than shown here due to its location in the low dielectric environment of the lipid membrane. B. View rotated 90° from A. Showing both the negatively charged binding site cleft (top) and positively charged cytoplasmic face (bottom). Poisson-Boltzmann electrostatics were calculated using the program APBS (Baker et al., Proc Natl Acad Sci USA, 98, 10037 (2001)) as implemented in Pymol (The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org). Pymol was used exclusively in the preparation of all figures.

FIG. 12. Comparison of the extracellular sides of β₂AR-T4L and rhodopsin. A. The N-terminus is missing from the experimental density in the β₂AR-T4L structure and is not shown. ECL2 is shown in green and contains a short a-helix and two disulfide bonds (yellow). The intraloop disulfide bond constrains the tip of ECL2 which interacts with ECL1. The second disulfide bond links ECL2 with helix III. There is one interaction between ECL2 and carazolol (blue) through Phe193^(5.32). The entire loop is held out of the ligand binding site by a combination of the rigid helical segment and the two disulfide bonds. B. In contrast, ECL2 (green) in rhodopsin assumes a lower position in the structure that occludes direct access to the retinal-binding site and forms a small β-sheet in combination with the N-terminal region (magenta) directly above the bound retinal (pink).

FIG. 13. Ligand binding characterization and comparison to rhodopsin. A. A view looking down on the plane of the membrane from the extracellular surface showing a detailed representation of the carazolol binding site in β₂AR-T4L. Carazolol is shown as sticks with carbon atoms colored yellow. β₂AR-T4L residues contributing to carazolol binding are shown in green and labeled. Electron density is contoured at 5a from an F_(o)-F_(c) omit map calculated without the contribution of carazolol. B. Binding orientation comparison between 11-cis-retinal in rhodopsin and carazolol in β₂AR-T4L. Van der Waals' surfaces for carazolol and retinal are represented as dots to accentuate the close packing interactions. Retinal in the all-cis conformation (pink), binds deep in the active site of rhodopsin as compared to carazolol (blue), packing its β-ionone ring between Tyr268^(6.51) and Phe212^(5.47) (cyan), blocking movement of Trp265^(6.48) (magenta) into the space. The β-ionone ring of trans-retinal in activated rhodopsin would not block Trp265^(6.48) from rotating into the space allowing a rotameric shift into its proposed active form. C. There are four residues involved in the toggle switch mechanism of β₂AR-T4L as shown. Phe290^(6.52) (magenta) is sandwiched between Phe208^(5.47) (tan) and Phe289^(6.51) (tan) forming a ring-face aromatic interaction. Like rhodopsin, an activation step is thought to occur by a rotameric change of Trp286^(6.48) (magenta) which would displace Phe290^(6.52). Carazolol is shown to interact extensively with the sandwich motif as shown: however, few interactions are seen with Trp286^(6.48). The 6.52 position in β₂AR-T4L is occupied by Phe290^(6.52) as opposed to Ala269^(6.52) in rhodopsin where the β-ionone ring replaces an aromatic protein side chain in forming the sandwich interactions. The aromatic character of the sandwich is otherwise maintained by Phe289^(6.51) and Phe208^(5.47) in β₂AR-T4L.

FIG. 14. Comparison of β₂AR-T4L helical orientations with rhodopsin (PDB ID Code 1U19). A. β₂AR-T4L is rendered as a ribbon trace colored with a blue to red spectrum corresponding to observed distances between Ca positions in the two structures (RMSD 2.7 Å between all residues in the transmembrane region). Helix II shows very little movement, whereas the entire lengths of helices III, IV, V shift significantly. Helix VIII and loops were not included in the comparison and are colored in tan. B. Movements of helices I and V of rhodopsin (grey) are shown relative to β₂AR-T4L. C. Movements of helices III, IV and VI. D. Ligand binding site representation. Carazolol is shown with yellow carbons. Entire helices are assigned a single designation based on their divergence from the rhodopsin position in the area of the ligand binding site as shown. Helix I is highly divergent, Helices H and VI are similar to rhodopsin. Helices IV and VII are moderately constant. Helices III and V are moderately divergent.

FIG. 15. Affinity curves for adrenergic ligands binding to β₂AR-T4L and wildtype β₂AR. Saturation curves for the antagonist [³H]DHA is shown at left, next to competition binding curves for the natural ligand (−)-Epinephrine and the high-affinity synthetic agonist Formoterol. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described above.

FIG. 16. Comparison of the proteolytic stability between the wild-type β₂AR and β₂AR-T4L in a limited trypsin proteolysis assay. TPCK-trypsin was added to carazolol-bound, purified, dodecylmaltoside-solubilized receptor at a 1:1000 ratio (wt:wt), and samples were analyzed by SDS-PAGE. Intact β₂AR-T4L (56.7 kD) and FLAG-tagged wild-type β₂AR (47.4 kD) migrate similarly as ˜55 kD bands. Markers are Biorad low-range SDS-PAGE protein standards.

FIG. 17. Stability comparison of unliganded β₂AR365 and β₂AR-T4L. For dodecylmaltoside-solubilized receptor preparations, maintenance of the ability to specifically bind [³H]DHA after incubation at 37° C. is taken as a measure of stability.

FIG. 18. Superimposed Ca traces of the receptor component of β₂AR-T4L (in blue) and β₂AR365 (in yellow). Common modeled transmembrane helix regions 41-58, 67-87, 108-137, 147-164, 204-230, 267-291, 312-326, 332-339 were used in the superposition by the program Lsqkab (The CCP4 Suite, Acta Crystallogr D Biol Crystallogr 50, 760 (1994)) (RMSD=0.8 Å).

FIG. 19. Carazolol dissociation from β₂AR365. Dodecylmaltoside-solubilized carazolol-bound receptor (at 50 μM) was dialyzed in a large volume of buffer containing 300 micromolar alprenonol as a competing ligand, and aliquots were removed from the dialysis cassette at different time points. Remaining bound carazolol was measured (in a relative sense) by collecting fluorescence emission with excitation at 330 nm and emission from 335 to 400 nm. For each carazolol fluorescence measurement, data was normalized for the protein concentration in the dialysis cassette (measured with the Bio-Rad Protein DC kit). The Y-axis represents carazolol fluorescence emission Intensity (in cps) at 341 nm. The exponential decay of carazolol concentration in the receptor dialysis cassette was fit using Graphpad Prism software, giving a half-life of 30.4 hrs.

FIG. 20. Comparison of β₁ and β₂AR sequences. After aligning the β₁ and β₂AR sequences, positions that have different amino acids between the two receptors were mapped onto the high-resolution structure of β₂ART4L (shown as red sticks). The carazolol ligand is shown as green sticks (with nitrogens in blue and oxygens in red). Highlighted residues Ala85^(2.56), Ala92^(2.63) and Tyr308^(7.35) are homologous to amino acids Leu110^(2.56), Thr117^(2.63) and Phe359^(7.35) of the β₁AR, which were shown to be primarily responsible for its selectivity over β₂AR for the compound RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)). In the β₂AR-T4L structure, only Tyr308^(7.35) faces the ligand, while Ala85^(2.56) lies at the interface between helices II and III. Of all the divergent amino acids, only Tyr308^(7.35) is found within 4 Å of any atom of carazolol.

FIG. 21. Design and optimization of the β₂AR-T4L fusion protein A. The sequence of the region of the β₂AR targeted for insertion of a crystallizable domain is shown (SEQ ID NO:2), and the positions of the junctions between the receptor and T4L (in red) for various constructs are indicated. The sequences that were initially replaced or removed are faded. Red lines are shown after every tenth residue. Peptide ‘LNKYADWT’ disclosed as SEQ ID NO: 3. B. Immunofluorescence images of HEK293 cells expressing selected fusion constructs. Panels on the left shows M1 anti-FLAG signal corresponding to antibody bound to the N-terminus of the receptor. Panels on the right show the same signal merged with blue emission from DAPI (nuclear staining for all cells). Plasma membrane staining is observed in the positive control, D3 and D1, while C3 and D5 are retained in the endoplasmic reticulum.

FIG. 22. Functional characterization of β₂AR-T4L. A. Affinity competition curves for adrenergic ligands binding to β₂AR-T4L and wild-type β₂AR. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described in the methods section of Example 4. B. β₂AR-T4L is still able to undergo ligand-induced conformational changes. Bimane fluorescence spectra (excitation at 350 nm) of detergent-solubilized β₂AR-T4L and wild-type β₂AR truncated at 365, labeled under conditions that selectively modify Cys265^(6.27) (see methods section of Example 4), were measured after incubating unliganded receptor with compounds for 15 min at room temperature. The cartoon illustrates that the observed changes in fluorescence can be interpreted as a movement of the bimane probe from a more buried, hydrophobic environment to a more polar, solvent-exposed position.

FIG. 23. A. Side-by-side comparison of the crystal structures of the β₂AR-T4L fusion protein and the complex between β₂AR365 and a Fab fragment. The receptor component of the fusion protein is shown as a blue cartoon (with modeled carazolol as red spheres), while the receptor bound to FabS is in yellow. B. Differences in the environment surrounding Phe264^(6.26) (shown as spheres) for the two proteins. C. The analogous interactions to the “ionic lock” between the E(D)RY motif and Glu247^(6.30) seen in rhodopsin (right panel, darkened) are broken in both structures of the β₂AR (left panel). Pymol (W. L. DeLano, The PyMOL Molecular Graphics System (2002) on the World Wide Web http://www.pymol.org) was used for the preparation of all figures.

FIG. 24. Schematic representation of the interactions between β₂AR-T4L and carazolol at the ligand binding pocket. Residues shown have at least one atom within 4 Å of the ligand in the 2.4 Å resolution crystal structure.

FIG. 25. The ligand binding pocket of β₂AR-T4L with carazolol bound. A. Residues within 4 Å of the ligand are shown as sticks, with the exception of A200, N293, F289, and Y308. Residues that form polar contacts with the ligand (distance cutoff 3.5 Å) are in green, other residues are gray (in all panels, oxygens are colored red and nitrogens are blue). B. Same as panel A, except that the ligand is oriented with its amine facing out of the page. W109 is not shown. C. Packing interactions between carazolol and all residues within 5 Å of the ligand. View is from the extracellular side of the membrane. Carazolol is shown as yellow spheres, receptor residues are shown as sticks within van der Waals dot surfaces. D. Model of (−)-isoproterenol (magenta sticks) in the ligand binding pocket observed in the crystal structure. A model of the agonist with optimal bond lengths and angles was obtained from the PRODRG server (Schuettelkopf, et al., Acta Crystallogr D Biol Crystallogr D60, 1355 (2004)), and the dihedral angles were adjusted to the values observed in the homologous atoms of bound carazolol (16-22 in FIG. 24). The one remaining unaccounted dihedral in (−)-isoproterenol was adjusted in order to place the catechol ring in the same plane as the C₁₆-C₁₅-O₁₄ plane in carazolol. Residues known to specifically interact with agonists are shown as green sticks.

FIG. 26. Packing interactions in the β₂AR that are likely to be modulated during the activation process. A. On the left, residues previously demonstrated to be CAMs (Rasmussen et al., Mol Pharmacol 56, 175 (1999); Tao, et. al., Mol Endocrinol 14, 1272 (2000); Jensen et al., J Biol Chem 276, 9279 (2001); Shi et al., J Biol Chem 277, 40989 (2002); Zuscik, et. al., (1998)) or UCMs (Strader et al., Proc Natl Acad Sci USA 84, 4384 (1987); Chung, et al., J Biol Chem 263, 4052 (1988); Moro, et. al., J Biol Chem 269, 6651 (1994); Green, et. al., J Biol Chem 268, 23116 (1993); Gabilondo et al., Proc Natl Acad Sci USA 94, 12285 (1997)) are shown as van der Waals spheres mapped onto a backbone cartoon of the β₂AR-T4L structure. On the right, residues that are found within 4 Å of the CAMs Leu124^(3.43) and Leu272^(6.34) are shown as yellow spheres or dot surfaces. A vertical cross-section through the structure illustrates that these surrounding residues connect the CAMs on helices III and VI with the UCMs on helix VII through packing interactions. B. In both β₂AR-T4L (blue) and rhodopsin (purple), a network of ordered water molecules is found at the interface between the transmembrane helices at their cytoplasmic ends. C. Network of hydrogen bonding interactions between water molecules and β₂AR-T4L residues (sidechains as blue sticks), notably the UCMs on helix VII (orange cartoon).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides methods and compositions for generating crystal structures of membrane proteins that diffract to resolutions as low as 2 to 3 Ångstroms. In one embodiment, the methods involve the crystallization of proteins in a lipidic cubic phase, wherein the host lipid comprises an additive, e.g., a sterol, such as cholesterol. The invention also provides the crystallized membrane proteins themselves, wherein the crystallized membrane proteins include GPCRs or modified GPCRs. The crystallized proteins can also include bound ligands, natural agonists, antagonists, and/or allosteric effectors. The invention additionally provides methods of using the 3-dimensional structures of the proteins (obtained from the crystals) to screen for novel ligands, drugs, and other useful molecules that affect the conformation and/or activity of the proteins in vitro or in vivo.

More specifically, the invention provides particular crystal forms of GPCRs diffracting to high resolutions. GPCRs have been grouped into five classes (Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based on sequence conservation, with class A GPCRs, including β₂AR, being the largest and most studied. β₂AR agonists are used in the treatment of asthma and preterm labor (DeLano, The PyMOL Molecular Graphics System (2002) on World Wide Web at pymol.org). The crystal forms provided by the invention include several diffraction-quality class A GPCR crystals, including crystals comprising β2AR and crystals comprising the human adenosine A_(2A) receptor.

The invention provides a three-dimensional structure of a human β₂AR protein comprising a T4-lysozyme (T4L) in place of the third intracellular loop (“β₂AR-T4L”) that has been solved in the presence of carazolol (2-propanol, 1-(9H-carbazol-4-yloxy)-3-[(1-methylethyl)amino] at 2.4 Å resolution. Additional class A GPCR structures make it possible to correlate sequence differences between GPCRs, e.g., between rhodopsin and β2AR, with emperically determined structural differences and extrapolate to other class A GPCRs. Highlighting interactions that constrain class A receptors into each of the two observed states allows a more comprehensive analysis of structural divergence and, therefore, more accurate models. Furthermore, GPCR structures provide an alternative signaling state on which to base homology models that will be more relevant for virtual ligand screening and structure-based drug design (Bissantz, et. al, Proteins 50, 5 (2003); Gouldson et al., Proteins 56, 67 (2004)).

DEFINITIONS

Terms used in the claims and specification are defined as set forth below unless otherwise specified. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

As used herein, the term “binding site” or “binding pocket” refers to a region of a protein that binds or interacts with a particular compound.

As used herein, the terms “binding” or “interaction” refers to a condition of proximity between a chemical entity, compound, or portions thereof, with another chemical entity, compound or portion thereof. The association or interaction can be non-covalent—wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions—or it can be covalent.

As used herein, the term “residue” refers to an amino acid that is joined to another by a peptide bond. Residue is referred to herein to describe both an amino acid and its position in a polypeptide sequence.

As used herein, the term “surface residue” refers to a residue located on a surface of a polypeptide. In contrast, a buried residue is a residue that is not located on the surface of a polypeptide. A surface residue usually includes a hydrophilic side chain. Operationally, a surface residue can be identified computationally from a structural model of a polypeptide as a residue that contacts a sphere of hydration rolled over the surface of the molecular structure. A surface residue also can be identified experimentally through the use of deuterium exchange studies, or accessibility to various labeling reagents such as, e.g., hydrophilic alkylating agents.

As used herein, the term “polypeptide” refers to a single linear chain of 2 or more amino acids. A protein is an example of a polypeptide.

As used herein, the term “homolog” refers to a gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, can apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication.

As used herein, the term “conservation” refers to a high degree of similarity in the primary or secondary structure of molecules between homologs. This similarity is thought to confer functional importance to a conserved region of the molecule. In reference to an individual residue or amino acid, conservation is used to refer to a computed likelihood of substitution or deletion based on comparison with homologous molecules.

As used herein, the term “distance matrix” refers to the method used to present the results of the calculation of an optimal pairwise alignment score. The matrix field (i,j) is the score assigned to the optimal alignment between two residues (up to a total of i by j residues) from the input sequences. Each entry is calculated from the top-left neighboring entries by way of a recursive equation.

As used herein, the term “substitution matrix” refers to a matrix that defines scores for amino acid substitutions, reflecting the similarity of physicochemical properties, and observed substitution frequencies. These matrices are the foundation of statistical techniques for finding alignments.

As used herein, the term “pharmacophore” refers to an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger or block a biological response. A pharmacophore can be used to design one or more candidate compounds that comprise all or most of the ensemble of steric and electronic features present in the pharmacophore and that are expected to bind to a site and trigger or block a biological response.

As used herein, the term “G-protein coupled receptor” (or “GPCR”) refers to a member of a family of heterotrimeric guanine-nucleotide binding protein (“G-protein”) coupled receptors (Pierce, et al., Nat. Rev. Mol. Cell. Biol. 3:630 (2002)). GPCRs share a common structural signature of seven membrane-spanning helices with an extra-cellular N terminus and an intracellular C terminus. The family has been grouped into at least five classes (designated A, B, C, D, E, etc.; see, e.g., Fredriksson, et al., Mol Pharmacol 63, 1256 (2003)) based on sequence conservation. When used without a descriptive limitation, the term “a G-protein couple receptor” includes GPCRs with native amino acid sequences as well as genetically engineered or otherwise mutated GPCR proteins. Mutated GPCR proteins include those comprising point mutations, truncations, inserted sequences or other chemical modifications, while retaining ligand binding activity. One example of a GPCR referred to herein that comprises a point mutation is β2AR^(E122W). An example of a GPCR referred to herein that comprises an inserted T4 lysozyme sequence is the human A_(2a) adenosine receptor-T4L.

Adrenergic receptors in the class A or amine group are some of the most thoroughly investigated GPCRs (Kobilka, Annu Rev Neurosci 15, 87 (1992); Caron, et al., Recent Prog Horm Res 48, 277 (1993); Strosberg, Protein Sci 2, 1198 (1993); Hein, et al., Trends Cardiovasc Med 7, 137 (1997); Rohrer, J Mol Med 76, 764 (1998); Xiang, et al., Adrenergic Receptors, 267 (2006)), and are composed of two main subfamilies, α and β, which differ in tissue localization and ligand specificity, as well as in G protein coupling and downstream effector mechanisms (Milligan, et al., Biochem Pharmacol 48, 1059 (1994)). Some representative class A receptors include the human A_(2A) adenosine receptor and the beta-2 adrenergic receptor. The term “beta-2 adrenergic receptor” (or “β₂AR” or “β2AR”) refers to a class A GPCR that responds to diffusable hormones and neurotransmitters and resides predominantly in smooth muscles throughout the body. When used without a descriptive limitation, the term “β2AR” includes β2ARs with native amino acid sequences as well as genetically engineered or otherwise mutated β2AR proteins. Mutated β2AR proteins include those comprising point mutations, truncations, inserted sequences or other chemical modifications, while retaining ligand binding activity. One example of a β2AR referred to herein that comprises a point mutation is β2AR^(E122W). An example of a β2AR referred to herein that comprises an inserted T4 lysozyme sequence is the human adenosine receptor β2AR^(E122W)-T4L.

The term “diffracts to a resolution of xx-yy Angstroms” means that diffraction data exceeding a predetermined signal to noise ratio can be obtained within the stated resolution range. In some embodiments, that diffraction data can be obtained using synchrotron radiation. Also, in some embodiments, that diffraction data can be obtained following freezing of the crystal in liquid nitrogen.

As used herein, the term “atomic co-ordinates” refers to a set of three-dimensional co-ordinates for atoms within a molecular structure. In one embodiment, atomic-co-ordinates are obtained using X-ray crystallography according to methods well-known to those of ordinarily skill in the art of biophysics. Briefly described, X-ray diffraction patterns can be obtained by diffracting X-rays off a crystal. The diffraction data are used to calculate an electron density map of the unit cell comprising the crystal; said maps are used to establish the positions of the atoms (i.e., the atomic co-ordinates) within the unit cell. Those of skill in the art understand that a set of structure co-ordinates determined by X-ray crystallography contains standard errors. In other embodiments, atomic co-ordinates can be obtained using other experimental biophysical structure determination methods that can include electron diffraction (also known as electron crystallography) and nuclear magnetic resonance (NMR) methods. In yet other embodiments, atomic co-ordinates can be obtained using molecular modeling tools which can be based on one or more of ab initio protein folding algorithms, energy minimization, and homology-based modeling. These techniques are well known to persons of ordinary skill in the biophysical and bioinformatic arts, and are described in greater detail below.

Atomic co-ordinates for binding pockets, such as, e.g., the ligand binding pocket of β2AR, and/or other agonist/antagonist binding sites of the present invention are intended to encompass those co-ordinates set out in the .pdb file (Appendix I; SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) incorporated into this specification, as well as co-ordinates that are substantially equivalent. Substantially equivalent co-ordinates are those that can be related to a reference set of co-ordinates by transformation reflecting differences in the choice of origin or inter-axis angels for one or more axes used to define the coordinate system. Operationally, co-ordinates are “substantially equivalent” when the structures represented by those co-ordinates can be superimposed in a manner such that root mean square deviations (RMSD) of atomic positions for the structures differs by less than a predetermined threshold. In some embodiments that threshold is less than about 5 Angstroms, or less than about 4 Angstroms, or less than about 3 Angstroms, or less than about 2 Angstroms, or less than about 1 Angstrom, or less than about 0.9 Angstrom, or less than about 0.8 Angstrom, or less than about 0.7 Angstrom, or less than about 0.6 Angstrom, or less than about 0.5 Angstrom, or less than about 0.4 Angstrom, or less than about 0.3 Angstrom. Preferably, co-ordinates are considered “substantially equivalent” when the RMSD is less than about 1 Angstrom. Methods for structure superpositioning and RMSD calculations are well known to those of ordinary skill in the art, and can be carried out using programs such as, e.g., the programs listed in Table 5 below.

Structural similarity can be inferred from, e.g., sequence similarity, which can be determined by one of ordinary skill through visual inspection and comparison of the sequences, or through the use of well-known alignment software programs such as CLUSTAL (Wilbur et al., J. Proc. Natl. Acad. Sci. USA, 80, 726 730 (1983)) or CLUSTALW (Thompson et al., Nucleic Acids Research, 22:4673 4680 (1994)) or BLAST® (Altschul et al., J. Mol. Biol., October 5; 215(3):403 10 (1990)), a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. CLUSTAL W is available at the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/); BLAST is available from the National Center for Biotechnology website (http://www.ncbi.nlm.nih.gov/BLAST/). A residue within a first protein or nucleic acid sequence corresponds to a residue within a second protein or nucleic acid sequence if the two residues occupy the same position when the first and second sequences are aligned.

The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence co-ordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI web-site).

The term “sterol” refers to a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring. See Fahy E. Subramaniam S et al., “A comprehensive classification system for lipids,” J. Lipid Res. 46 (5):839-861 (2005)). Sterols are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. Sterols can include, e.g., cholesterol or cholesteryl hemisuccinate (“CHS”).

The term “atomic co-ordinates for residues” refers to co-ordinates for all atoms associated with a residue, or for some of the atoms such as, e.g., side chain atoms.

The term “atomic co-ordinates of a candidate compound” refers to co-ordinates for all atoms comprising the compound or a subset of atoms comprising the compound.

The term “characterizing a binding interaction” refers to characterizing any observable property of a first molecule and determining an whether there is a change in that observable property after contacting the first molecule with a second molecule under conditions in which said first and second molecules can potentially bind.

The term “antagonist” refers to molecules that bind to and block the active site of a protein, but do not affect the equilibrium between inactive and active states. In contrast, an “agonist” is a ligand that shifts the equilibrium to an active receptor state. An “inverse agonist” is a ligand that acts to reduce the basal activity of a receptor through interactions that shift the equilibrium to more of an inactive state.

Ballesteros-Weinstein numbering appears in the text and Figures as superscripts to the protein numbering. Within each helix is a single most conserved residue among the class A GPCRs. This residue is designated X.50, where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Method for Producing Diffraction Quality Crystals of Membrane Proteins

In one aspect, the present invention discloses a modified lipidic cubic mesophase method for crystallizing proteins (see, e.g., Cherezov et al., Biophysical J., v.83, 3393-3407 (2002)). The novel method described herein yields diffraction quality crystals of membrane proteins and is particularly useful for generating crystals of G-protein coupled receptor proteins (“GPCRs”). The method has now been successfully applied to diverse members of this important family of proteins, yielding crystals that diffract to resolutions in the 2.5 Å range. Among other advantages, this method allows diffraction-quality crystals of membrane proteins to be generated in the absence of any stabilizing antibodies bound to the protein in the crystal.

The LCP/sterol crystallization method described herein includes a step of mixing a solution containing the protein of interest with a host lipid or a host lipid mixture that includes a lipid additive. Given the teaching provided herein, one skilled in the art will recognize that a variety of host lipids may suffice for the generation of a cubic mesophase, e.g., hydrated monounsaturated monoacylglycerols such as monoolein, monopalmitolein, and/or monovacennin. The host lipid 1-monoolein is a preferred host lipid for certain applications of the method. In embodiments utilizing a lipid mixture, a lipid additive that is distinct from the host lipid is included, e.g., monounsaturated monoacylglycerols or other hydrophobic molecules known to interact with membranes or membrane-associated proteins such as, 2-monoolein, phosphotidylcholine, cardiolipin, lyso-PC, a polyethylene glyocol-lipid, dioleoylphosphatidylethanolamine (“DOPE”), DOPE-Me, dioleoyl phosphatidylcholine (“DOPC”), Asolectin, or a sterol (e.g., cholesterol, ergosterol, etc.). An example of a lipid mixture for GPCR crystallization is one comprising cholesterol as lipid additive in a ratio between 1 and 50% w/w relative to the host lipid, more preferably between 5 and 20%, and even more preferably between 8 and 12%. The protein mixture may include ligands of physiological interest and/or ligands that stabilize the protein. In, the case of GPCRs, the ligands may include various agonists and antagonists known to the artisan, including well-known agonists such as carazolol (an inverse agonist), timolol, and other molecules including, without limitation, Examples of ligands, include but are not limited to carazolol, light and olfactory stimulatory molecules; adenosine, bombesin, bradykinin, endothelin, γ-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), glucagon, acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin releasing hormone (TRH), and oxytocin).

A typical concentration of protein in the protein mixture is 25-75 mgs/ml but this concentration may vary according to protein identity and purification methods. As will be recognized by the skilled artisan, the concentration must be high enough to result in a degree of insolubility sufficient for nucleation to occur after a precipitation solution is combined with the protein-laden lipid solution; on the other hand, concentrations of protein that are too high may prevent the orderly growth of high-quality crystals.

The lipid mixture is preferably combined with the protein mixture and homogenized, e.g., using a syringe mixer, spontaneously yielding a homogenous cubic phase. Typically, the lipid mixture is added to the protein solution at a ratio of 1:1, 3:2, 4:2 w/w lipid:protein, but this ratio may be varied by the skilled artisan as desired, depending on various parameters, e.g., the concentration of protein in the protein mixture. The protein-laden lipidic cubic phase preparation thus obtained is then combined with precipitation solution (also referred to as crystallization solution) on or in an appropriate surface or container, e.g., a glass sandwich plate with wells where the mixed solutions can incubate while crystallization occurs. A typical volume of the protein-laden lipidic cubic phase used in the method is between 10 and 100 mL, with 40 to 60 mL preferred in certain embodiments. A typical volume of precipitation solution is 20 to 100 times greater, e.g., for a 20 mL volume of protein-laden lipidic cubic phase, approximately 1 μL of precipitation solution would be added to initiate crystallization.

The precipitation solution used in the crystallization method is an appropriately buffered solution (i.e., buffered to approximate the physiological conditions of the native protein) comprising polyethylene glycol, a salt, and optionally a small soluble molecule such as an alcohol.

With respect to the polyethylene glycol in the precipitation solution, useful PEG molecules include PEG 300, PEG 400, PEG 550, PEG 550mme, PEG 1000, and PEG 1500, as well as other PEG molecules with average molecular weights less than 2000. In certain embodiments, larger average molecular weight PEG molecules (up to 20,000) or modified PEG molecules may be preferred. In some embodiments, the PEG or modified PEG has an average molecular weight of 400. Examples of modified PEG include but are not limited to PEG laurate, PEG dilaurate, PEG oleate, PEG dioleate, PEG stearate, PEG distearate, PEG glyceryl trioleate, PEG glyceryl laurate, PEG glyceryl stearate, PEG glyceryl oleate, PEG palm kernel oil, PEG hydrogenated castor oil, PEG castor oil, PEGcom oil, PEG caprate/caprylate glycerides, PEG caprate/caprylate glycerides, PEG cholesterol, PEG phyto sterol, PEG soya sterol, PEG trioleate, PEG sorbitan oleate, PEG sorbitan laurate, PEG succinate, PEG nonyl phenol series, PEG octyl phenol series, Methyl-PEG, PEG-Maleimide, PEG4-NHS Ester and methoxypoly(ethylene glycol) (mPEG).

PEG may be present in the crystallization solution in concentrations between 10-60% v/v, and most typically between 20-40% v/v. The preferred concentration will vary depending on the average molecular weight of PEG utilized, i.e., 10-60% v/v of PEG will be preferred for PEG≦1000 whereas 10-30% w/v will be preferred for PEG>1000 (larger average molecular weight PEG formulations are described in % w/v rather than % v/v).

With respect to the salt used in the method, an optimum cation can usually be found for a given crystal. Both sodium and lithium sulfate have proven useful for obtaining high resolution proteins of GPCRs. Again, the concentrations may be varied up to 1 M, with lower concentrations of approximately 50-200 mM typically preferred. Other organic salts, e.g., citrate, malonate, tartrate, formate and acetate, may also be screened for their effects on crystal formation. In certain embodiments, the precipitation solution additionally comprises a small organic molecule such as an alcohol, a diol or a triol, e.g., a hexanediol, a butanediol, or derivative thereof. These molecules may be present in the precipitation solution in various concentrations, as appropriate, but typically in the range of 1-20% v/v, more typically in the 5-10% v/v range. In certain embodiments, preferred combinations of lipid additives (in the protein-laden lipidic cubic phase mixture) and small molecules (in the precipitation solution) yield optimal results. Examples of such combinations include 1,4-butanediol in combination with DOPE or cholesterol, and 2,6-hexanediol in combination with cholesterol.

In optimizing the conditions from micro-crystals to larger crystals for a given system (e.g., a protein/ligand system), the choice and concentration of a specific sterol(s) and specific lipid(s), as well the pH, buffer system, salt, and salt concentration may be varied, as in other types of crystallization formats. As noted above, small organic additives, especially alcohols and diols such as 1,4 butanediol, 1,6 hexanediol, etc., can be particularly useful in generating large diffraction quality crystals. Also, due to the membrane fluidity-altering properties of cholesterol and other sterols, sterol and precipitant concentration should be treated as dependent variables. For example, increasing concentrations of cholesterol in monoolein serve to rigidify the membranes, potentially slowing diffusion of the membrane protein within the lipid matrix. Conversely, increasing concentrations of PEG 400 swell the cubic phase, thereby increasing the lattice parameter of the matrix and speeding diffusion within the lipid. The former scenario would slow the rate of crystallization while the latter would increase the rate. The two effects should therefore be balanced for optimal nucleation and also for optimal growth of large, well-ordered crystals that diffract to a high resolution.

The mixing of the protein-laden lipidic cubic phase solution and the precipitation solution typically occurs at room temperatures. After set-up, the plates containing the mixed crystallization solutions can be monitored as often as desired for the appearance of crystal growth. One skilled in the art will recognize that further optimization of these conditions may be desirable, for example, to maximize the size and number of diffraction quality crystals that are obtained. In making determinations as to the preferred molecules and conditions for crystallization, the skilled artisan may rely on well-known phase diagrams and other previously determined physical constants, in addition to the novel methodology and Examples described herein. For certain lipid mixtures, pre-screening their phase behavior by microscope visualization and/or by X-ray prior to being mixed with the protein solution may facilitate the process of optimization. An in meso crystallization robot and automatic imager combined with multiple 96-well optimization screens can be used to run thousands of trials in a relatively facile manner.

It also possible to achieve additional stabilization of proteins and improve the yield of diffraction-quality crystals using the LCP/sterol method described herein by modification of the protein. For example, an unstable region of the protein may be replaced or stabilized by incorporation of a portion of a stable protein, e.g., a T4 lysozyme, whose structure is previously known but which does not (when fused) significantly affect the biochemical activity of the protein of interest. For example, the ECL2 and ECL3 regions of a PAR can be stabilized by such modifications, as described herein (Examples 3 and 4). Other modifications include one or more point mutations that do not significantly alter the properties of the protein of interest except to increase its stability and/or tendency to crystallize well. For example, β2AR^((E122W)) comprises an E122W point mutation and yields crystals with the LPC/sterol method. Analogous residues in other GPCRs could be modified in the same way. One advantage of the LCP method applied to both modified and unmodified proteins, as noted above, is that it allows (but does not preclude) the crystallization of proteins in the absence of heterologous proteins, such as antibodies, that may not be of interest to the crystallographer.

Method of Ligand Screening by Lipidic Cubic Phase Crystallization

Aspects of the lipidic cubic phase crystallization methodology described above can be modified for the purpose of determining low affinity lipid binding sites within integral membrane proteins through co-crystallization trials within a lipidic cubic phase matrix. In this method various lipids of different composition are incorporated at a variety of concentrations into monoolein, wherein the monoolein is solubilized in chloroform or heated to its fluid isotropic phase. Crystal growth is then assessed by visual inspections and diffraction data collected on any crystalline material within the experiment. Because the lipid is low affinity the method requires an environment conducive for free exchange of lipid from annular to non-annular protein binding sites. The presence of interpretable electron density not associated with crystal packing interfaces allows the inference of specific binding sites for a particular lipid within the context of the membrane protein in a membrane environment. Because the binding occurs within a membrane the complicating factor of detergent partitioning is eliminated and thermodynamics of association are more realistic. The method thus allows one to characterize in detail previously inaccessible regions of membrane proteins, as well as describe and exploit binding interactions that might otherwise remain undetected. Furthermore, the technique can be applied to ligand binding studies where the ligand occupies a site on the membrane protein that is juxtaposed to the lipid plane relying on partitioning into the aqueous phase to enable saturation of the site. This limits the exposure of the protein to harsh organic co-solvents and may also find utility for soluble proteins that crystallize within the lipidic cubic phase solvent channels.

By wan of example, existing crystallization conditions for a protein can be utilized as a starting point for screening novel ligands to the β2-adrenergic receptor. In the first instance, cholesterol solubilized in chloroform can be incorporated into chloroform-solubilized monoolein at weight ratio of 10%. After drying and desiccating the mixture, protein at 30-80 mg/mL can incorporated at 2/3 volume ratio and used for crystallization trials. A similar protocol was used for other lipid like molecules, including cholesteryl hemisuccinate and a variety of other cholesterol analogs. In each case protein was incorporated into the resulting mixture and screened for crystallization. Binding of the novel ligand to the receptor is indicated by diffraction quality crystals and ultimately by three-dimensional structural data. By incorporating analogues of cholesterol we are able to map out the binding specificities based on the unique structural features of their respective sterol rings and polar moieties and, if their incorporation led to diffraction quality crystals, the interactions between the protein and cholesterol analogue are determined.

This method of ligand screening is not limited to lipid-like molecules, as we can use the lipidic cubic phase as a host for other highly hydrophobic molecules that act at orthosteric binding sites. One problem with structure based or fragment based design of novel ligands is the hydrophobicity often associated with potential drug leads. This is a problem in aqueous based crystallization schemes because the solubility of the ligand is often less than 1 mM and unless there is a slow off rate from the protein of interest the binding site will be in a ligand depleted state at crystallization conditions which often involve protein concentrations between 0.5 and 1 mM. One can attempt to co-solubilize the hydrophobic ligand in aqueous miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). However, these often interfere with the stability or crystallization properties of the protein and their usefulness is not general. Therefore, this method allows one to incorporate the hydrophobic ligand directly into the lipidic cubic phase where its accessibility to the protein will be limited by the partitioning between the lipid and aqueous phase and/or the accessibility of the binding.

Additional guidance relating to these methods is provided by the working and prophetic examples of protein crystallization presented herein.

The Crystal Structure of Human β2AR Bound to Carazolol and Uses Thereof

G-protein coupled receptors are cell surface receptors that indirectly transduce extracellular signals to downstream effectors, e.g., intracellular signaling proteins, enzymes, or channels. G-protein coupled receptor membrane proteins are grouped into one of at least 6 classes (i.e., A, B, C, D, E, and F). An example of a mammalian G-protein coupled receptor is the β2A receptor, a receptor in the Class A subfamily of GPCRs.

Class A GPCRs function in a variety of physiological processes such as vasodilation, bronchodilation, neurotransmitter signaling, stimulation of endocrine secretions, gut peristalsis, development, mitogenesis, cell proliferation, cell migration, immune system function, and oncogenesis. Accordingly, class A GPCRs can be used as screening targets to identify modulators of these processes which can then function to ameliorate diseases associated with these processes, e.g., cancer and autoimmunity.

The 2.4 Angstrom structure of β2AR bound to carazolol, described herein (PDB coordinates appear in Appendix I; SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) can be used as a model for rationally designing pharmacophore and/or candidate compounds, either de novo or by modification of known compounds. As noted below, the multiple ligand binding sites in this structure include amino acids that are highly conserved across a large number of class A G protein coupled receptors (GPCRs) indicating that the 2.4 Angstrom structure of β2AR can be used for the rational designing of ligands (e.g., therapeutic compounds) that bind to this receptor and others. Pharmacophore and candidate compounds identified through the use of the crystal structure co-ordinates will have utility as pharmaceuticals due to their ability to alter the structure and/or binding properties of β2AR. Pharmacophores and candidate compounds can be determined according to any method known in the art, including the methods described in U.S. Pat. No. 5,888,738 to Hendry, and the methods described in U.S. Pat. No. 5,856,116 to Wilson et al. the disclosures of which both are incorporated by reference in their entirety for all purposes.

The structure data provided herein can be used in conjunction with computer-modeling techniques to develop models of sites on the human β2AR or related GPCRs selected by analysis of the crystal structure data. The site models characterize the three-dimensional topography of site surface, as well as factors including van der Waals contacts, electrostatic interactions, and hydrogen-bonding opportunities. Computer simulation techniques can be used to map interaction positions for functional groups including protons, hydroxyl groups, amine groups, divalent cations, aromatic and aliphatic functional groups, amide groups, alcohol groups, etc. that are designed to interact with the model site. These groups can be designed into a pharmacophore or candidate compound with the expectation that the candidate compound will specifically bind to the site. Pharmacophore design thus involves a consideration of the ability of the candidate compounds falling within the pharmacophore to interact with a site through any or all of the available types of chemical interactions, including hydrogen bonding, van der Waals, electrostatic, and covalent interactions, although, in general, and preferably, pharmacophores interact with a site through non-covalent mechanisms.

The ability of a pharmacophore or candidate compound to bind to the human β2AR can be analyzed prior to actual synthesis using computer modeling techniques. Only those candidates that are indicated by computer modeling to bind the target with sufficient binding energy (i.e., binding energy corresponding to a dissociation constant with the target on the order of 10⁻² M or tighter) can be synthesized and tested for their ability to bind to the human β2AR using binding assays or functional assays known to those of skill in the art. The computational evaluation step thus avoids the unnecessary synthesis of compounds that are unlikely to bind β2AR or one or more of its constitutive binding sites, or the related binding sites of another GPCR with adequate affinity.

A human β2AR or candidate compound(s) can be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with individual binding target sites on β2AR or binding site thereof, including, but not limited to a binding pocket of the human β2AR. One skilled in the art can use one of several methods to screen chemical entities or fragments for their ability to associate with one or more of these human β2AR binding sites. For example, increased affinity and specificity may be designed into caffeine and other xanthine molecules by combining interactions with both xanthine and non-xanthine binding sites.

The process can begin by visual inspection of, for example a target site on a computer screen, based on the human β2AR co-ordinates, or a subset of those co-ordinates (e.g., binding pocket residues V 117, T118, F193, Y199, A200, W286, F289, F290, and Y316), as set forth in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). Selected fragments or chemical entities can then be positioned in a variety of orientations or “docked” within a target site of the human β2AR as defined from analysis of the crystal structure data. Docking can be accomplished using software such as Quanta (Molecular Simulations, Inc., San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields such as CHARMM (Molecular Simulations, Inc., San Diego, Calif.), ICM (Molsoft, San Diego, Calif.), and AMBER (University of California, San Francisco).

Specialized computer programs can also assist in the process of selecting fragments or chemical entities. These include but are not limited to: GRID (Goodford, P. J., “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules,” J. Med. Chem., 28, pp. 849 857 (1985)); GRID is available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method,” Proteins: Structure, Function and Genetics, 11, pp. 29 34 (1991)); MCSS is available from Molecular Simulations, Inc., San Diego, Calif.; AUTODOCK (Goodsell, D. S, and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing,” Proteins: Structure, Function, and Genetics, 8, pp. 195 202 (1990)); AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK (Kuntz, I. D., et al. “A Geometric Approach to Macromolecule-Ligand Interactions,” J. Mol. Biol., 161, pp. 269 288 (1982)); DOCK is available from University of California, San Francisco, Calif.; CERIUS H (available from Molecular Simulations, Inc., San Diego, Calif.); and Flexx (Raret, et al. J. Mol. Biol. 261, pp. 470 489 (1996)).

After selecting suitable chemical entities or fragments, they can be assembled into a single compound. Assembly can proceed by visual inspection of the relationship of the fragments to each other on a three-dimensional image of the fragments in relation to the human β2AR or its binding sites or those of a related GPCR receptor structure or portion thereof displayed on a computer screen. Visual inspection can be followed by manual model building using software such as the Quanta or Sybyl programs described above.

Software programs also can be used to aid one skilled in the art in connecting the individual chemical entities or fragments. These include, but are not limited to CAVEAT (Bartlett, P. A., et al. “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules” In “Molecular Recognition in Chemical and Biological Problems,” Special Publ, Royal Chem. Soc., 78, pp. 182 196 (1989)); CAVEAT is available from the University of California, Berkeley, Calif.; 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.); this area is reviewed in Martin, Y. C., “3D Database Searching in Drug Design,” J. Med. Chem., 35:2145 2154 (1992)); and HOOK (available from Molecular Simulations Inc., San Diego, Calif.).

As an alternative to building candidate pharmacophores or candidate compounds up from individual fragments or chemical entities, they can be designed de novo using the structure of the β2AR, its constituent ligand binding pocket, or the homologous cavities in a related GPCR, optionally, including information from co-factor(s) or known activators or inhibitor(s) that bind to the target site. De novo design can be implemented by programs including, but not limited to LUDI (Bohm, H. J., “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors,” J. Comp. Aid. Molec. Design, 6, pp. 61 78 (1992)); LUDI is available from Molecular Simulations, Inc., San Diego, Calif.; LEGEND (Nishibata, Y., and Itai, A., Tetrahedron 47, p. 8985 (1991); LEGEND is available from Molecular Simulations, San Diego, Calif.; and LeapFrog (available from Tripos Associates, St. Louis, Mo.).

The functional effects of known β2AR also can be altered through the use of the molecular modeling and design techniques described herein. This can be carried out by docking the structure of the known ligand on a human A_(2A) adenosine receptor or a model structure of one or more binding sites of the human β2AR (e.g., the binding pocket described herein) and modifying the shape and charge distribution of the ligand or protein model structure to optimize the binding interactions between the ligand and protein. The modified structure can be synthesized or obtained from a library of compounds and tested for its binding affinity and/or effect on ribosome function. Of course, where the crystal structure of a complex between a human β2AR (or subunit thereof) and a ligand is known, comparisons between said complex and the structures of the present invention can be made to gain additional information about alterations in human β2AR conformation that occur upon ligand binding. This information can be used in design of optimized ligands. Compounds that interfere or activate human β2AR function (e.g., by interacting with a binding pocket) are especially well suited for the docking, co-crystallization, and optimization applications of the present invention.

Additional molecular modeling techniques also can be employed in accordance with the invention. See, e.g., Cohen, N. C., et al. “Molecular Modeling Software and Methods for Medicinal Chemistry,” J. Med. Chem., 33, pp. 883 894 (1990); Hubbard, Roderick E., “Can drugs be designed?” Curr. Opin. Biotechnol. 8, pp. 696 700 (1997); and Afshar, et al. “Structure-Based and Combinatorial Search for New RNA-Binding Drugs,” Curr. Opin. Biotechnol. 10, pp. 59 63 (1999).

Following pharmacophore or candidate compound design or selection according to any of the above methods or other methods known to one skilled in the art, the efficiency with which a candidate compound falling within the pharmacophore definition binds to the human β2AR or its ligand binding site, or alternatively binds to a related GPCR or homologous portions thereof, can be tested and optimized using computational evaluation. A candidate compound can be optimized, e.g., so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site. These repulsive electrostatic interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions. It is preferred that the sum of all electrostatic interactions between the candidate compound and the human β2AR, including its ligand binding site when the candidate compound is bound to the target make a neutral or favorable contribution to the binding enthalpy or free energy.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include, but are not limited to Gaussian 92, revision C (Frisch, M. J., Gaussian, Inc., Pittsburgh, Pa. (1992)); AMBER, version 4.0 (Kollman, P. A., University of California at San Francisco, (1994)); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. (1994)); and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif. (1994)). These programs can be run, using, e.g., a Silicon Graphics workstation, Indigo, 02-R10000 or IBM RISC/6000 workstation model 550. Other hardware and software combinations can be used to carry out the above described functions, and are known to those of skill in the art. In general, the methods described herein, particularly computer-implemented methods, comprise a step of recording or storing data onto a medium, wherein the medium can include a computer-readable medium. Additionally, or alternatively, the methods comprise a step of reporting or communicating the data to a user of interest, e.g., an operator of the device and/or computer that is employed in the method; or the computer can perform an additional useful task, e.g., alert the operator of the computer that a function has been completed, upon completing one or more determining steps of the method.

Once a pharmacophore or candidate compound has been optimally selected or designed, as described above, substitutions can then be made in some of its atoms or side groups to improve or modify its binding properties. Generally, initial substitutions are conservative in that the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Components known in the art to alter conformation should be avoided in making substitutions. Substituted candidates can be analyzed for efficiency of fit to the human β2AR (or one or more binding sites of the human β2AR) using the same methods described above.

Assays

Any one of a number of assays of function known to those of skill in the art can be used to determine the biological activity of candidate compounds.

Candidate compound interaction with the human β2AR (or one or more binding sites of human β2AR) or to a related GPCR or portion thereof can be evaluated using direct binding assays including filter binding assays, such as are known to those skilled in the art. Binding assays can be modified to evaluate candidate compounds that competitively inhibit the binding of, e.g., known human β2AR binding compounds including xanthine and xanthine-based compounds such as theophylline, theobromine and caffeine. These and other assays are described in International Publication WO 00/69391, the entire disclosure of which is incorporated by reference in its entirety for all purposes. Methods of assaying for modulators of ligand binding and signal transduction include in vitro ligand binding assays using GPCRs, such as human β2AR (or one or more binding sites selected from the binding pockets I, II and III of the human β2AR), portions thereof such as the extracellular domain, or chimeric proteins comprising one or more domains of a GPCR, oocyte GPCR expression or tissue culture cell GPCR expression, either naturally occurring or recombinant; membrane expression of a GPCR, either naturally occurring or recombinant; tissue expression of a GPCR; expression of a GPCR in a transgenic animal, etc.

As noted above, GPCRs and their alleles and polymorphic variants are G-protein coupled receptors that participate in signal transduction and are associated with cellular function in a variety of cells, e.g., neurons, immune system cells, kidney, liver, colon, adipose, and other cells. The activity of GPCR polypeptides can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding, (e.g., radioactive ligand binding), second messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and the like. Such assays can be used to test for inhibitors and activators of a GPCR. In particular, the assays can be used to test for compounds that modulate natural ligand-induced GPCR activity, for example, by modulating the binding of the natural ligand to the receptor and/or by modulating the ability of the natural ligand to activate the receptor. Typically in such assays, the test compound is contacted with the GPCR in the presence of the natural ligand. The natural ligand can be added to the assay before, after, or concurrently with the test compound. The results of the assay, for example, the level of binding, calcium mobilization, etc. is then compared to the level in a control assay that comprises the GPCR and natural ligand in the absence of the test compound.

Screening assays of the invention are used to identify modulators that can be used as therapeutic agents, e.g., antagonists of GPCR activity. For example, carazolol is a known high-affinity inverse agonist of human β2AR.

The effects of test compounds upon the function of the GPCR polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the GPCRs and natural ligand-mediated GPCR activity. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca²⁺, IP₃ or cAMP.

For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature 10:349:117-27 (1991); Bourne et al., Nature 348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem. 67:653-92 (1998).

Modulators of GPCR activity are tested using GPCR polypeptides, either recombinant or naturally occurring. The protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring. For example, neurons, cells of the immune system, adipocytes, kidney cells, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein or others as generally known in the art. Signal transduction can also be examined in vitro with soluble or solid state reactions, using a chimeric molecule such as an extracellular domain of a receptor covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain covalently linked to the transmembrane and or cytoplasmic domain of a receptor. Furthermore, ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding.

Ligand binding to a human β2AR (or one or more binding sites thereof) or a chimeric protein derivative can be tested in a number of formats. For example, binding can be performed in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Typically, in an assay of the invention, the binding of the natural ligand to its receptor is measured in the presence of a candidate modulator. Alternatively, the binding of the candidate modulator can be measured in the presence of the natural ligand. Often, competitive assay that measure the ability of a compound to compete with binding of the natural ligand to the receptor are used. Binding can be measured by assessing GPCR activity or by other assays: binding can be tested by measuring e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape) changes, or changes in chromatographic or solubility properties.

Receptor-G-protein interactions can also be used to assay for modulators. For example, in the absence of GTP, binding of an activator such as the natural ligand will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for inhibitors. For example, a ligand can be added to the human β2AR and G protein in the absence of GTP to form a tight complex. Inhibitors can be identified by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the alpha subunit of the G protein from the other two G protein subunits serves as a criterion of activation.

An activated or inhibited G-protein will in turn alter the properties of downstream effectors such as proteins, enzymes, and channels. The classic examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G-protein, phospholipase C by G_(q) and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins. Downstream consequences such as generation of diacyl glycerol and IP₃ by phospholipase C, and in turn, for calcium mobilization e.g., by IP₃ can also be examined. Thus, modulators can be evaluated for the ability to stimulate or inhibit ligand-mediated downstream effects. In other examples, the ability of a modulator to activate a GPCR expressed in adipocytes in comparison to the ability of a natural ligand, can be determined using assays such as lipolysis (see, e.g., WO01/61359).

Activated GPCRs become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well). Thus, activators will promote the transfer of ³²P from gamma-labeled GTP to the receptor, which can be assayed with a scintillation counter. The phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G-proteins. The kinase/arrestin pathway plays a key role in the desensitization of many GPCR receptors. Modulators can therefore also be identified using assays involving beta-arrestin recruitment. Beta-arrestin serves as a regulatory protein that is distributed throughout the cytoplasm in unactivated cells. Ligand binding to an appropriate GPCR is associated with redistribution of beta-arrestin from the cytoplasm to the cell surface, where it associates with the GPCR. Thus, receptor activation and the effect of candidate modulators on ligand-induced receptor activation, can be assessed by monitoring beta-arrestin recruitment to the cell surface. This is frequently performed by transfecting a labeled beta-arrestin fusion protein (e.g., beta-arrestin-green fluorescent protein (GFP)) into cells and monitoring its distribution using confocal microscopy (see, e.g., Groarke et al., J. Biol. Chem. 274(33):23263-69 (1999)).

Receptor internalization assays can also be used to assess receptor function. Upon ligand binding, the G-protein coupled receptor—ligand complex is internalized from the plasma membrane by a clathrin-coated vesicular endocytic process; internalization motifs on the receptors bind to adaptor protein complexes and mediate the recruitment of the activated receptors into clathrin-coated pits and vesicles. Because only activated receptors are internalized, it is possible to detect ligand-receptor binding by determining the amount of internalized receptor. In one assay format, cells are transiently transfected with radiolabeled receptor and incubated for an appropriate period of time to allow for ligand binding and receptor internalization. Thereafter, surface-bound radioactivity is removed by washing with an acid solution, the cells are solubilized, and the amount of internalized radioactivity is calculated as a percentage of ligand binding. See, e.g., Vrecl et al., Mol. Endocrinol. 12:1818-29 (1988) and Conway et al., J. Cell Physiol. 189(3):341-55 (2001). In addition, receptor internalization approaches have allowed real-time optical measurements of GPCR interactions with other cellular components in living cells (see, e.g., Barak et al., Mol. Pharmacol. 51(2)177-84 (1997)). Modulators can be identified by comparing receptor internalization levels in control cells and cells contacted with candidate compounds. For example, candidate modulators the human β2AR are assayed by examining their effects on receptor internalization upon binding of the natural ligand.

Another technology that can be used to evaluate GPCR-protein interactions in living cells involves bioluminescence resonance energy transfer (BRET). A detailed discussion regarding BRET can be found in Kroeger et al., J. Biol. Chem., 276(16):12736-43 (2001).

Receptor-stimulated guanosine 5′-O-(γ-Thio)-Triphosphate ([³⁵S]GTPγS) binding to G-proteins can also be used as an assay for evaluating modulators of GPCRs. [³⁵S]GTPγS is a radiolabeled GTP analog that has a high affinity for all types of G-proteins, is available with a high specific activity and, although unstable in the unbound form, is not hydrolyzed when bound to the G-protein. Thus, it is possible to quantitatively assess ligand-bound receptor by comparing stimulated versus unstimulated [³⁵S]GTPγS binding utilizing, for example, a liquid scintillation counter. Inhibitors of the receptor-ligand interactions would result in decreased [³⁵S]GTPγS binding. Descriptions of [³⁵S]GTPγS binding assays are provided in Traynor and Nahorski, Mol. Pharmacol. 47(4):848-54 (1995) and Bohn et al., Nature 408:720-23 (2000).

The ability of modulators to affect ligand-induced ion flux can also be determined. Ion flux can be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing a GPCR. One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard methodology (see, e.g., Hamil et al., Pfügers. Archie. 391:85 (1981). Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70 (1994)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.

Preferred assays for G-protein coupled receptors include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G-protein coupled receptors and the natural ligands disclosed herein as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage are monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that can be employed are those disclosed in the Molecular Probes 1997 Catalog. For G-protein coupled receptors, promiscuous G-proteins such as Ga15 and Ga16 can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053 (1991)). Such promiscuous G-proteins allow coupling of a wide range of receptors to signal transduction pathways in heterologous cells.

Receptor activation by ligand binding typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP₃, which releases intracellular stores of calcium ions. Activation of some G-protein coupled receptors stimulates the formation of inositol triphosphate (IP₃) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)). IP₃ in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP₃ can be used to assess G-protein coupled receptor function. Cells expressing such G-protein coupled receptors can exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it can be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.

Other assays can involve determining the activity of receptors which, when activated by ligand binding, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting downstream effectors such as adenylate cyclase. There are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci. U.S.A. 88:9868-9872 (1991) and Dhallan et al., Nature 347:184-187 (1990)). In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it can be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.

In one embodiment, changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995) can be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994) can be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with ³H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates are separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist to cpm in the presence of buffer control (which can or can not contain an agonist).

In another embodiment, transcription levels can be measured to assess the effects of a test compound on ligand-induced signal transduction. A host cell containing the protein of interest is contacted with a test compound in the presence of the natural ligand for a sufficient time to effect any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions can be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription can be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest can be detected using northern blots or their polypeptide products can be identified using immunoassays. Alternatively, transcription based assays using reporter genes can be used as described in U.S. Pat. No. 5,436,128, herein incorporated by reference. The reporter genes can be, e.g., chloramphenicol acetyltransferase, firefly luciferase, bacterial luciferase, beta-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it can be compared with the amount of transcription in a substantially identical cell that lacks the protein of interest. A substantially identical cell can be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the protein of interest.

Samples that are treated-with a potential GPCR inhibitor or activator are compared to control samples comprising the natural ligand without the test compound to examine the extent of modulation. Control samples (untreated with activators or inhibitors) are assigned a relative GPCR activity value of 100. Inhibition of a GPCR is achieved when the GPCR activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of a GPCR is achieved when the GPCR activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.

In one embodiment the invention provides soluble assays using molecules such as a domain, e.g., a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a GPCR; or a cell or tissue expressing a GPCR, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, GPCR, or cell or tissue expressing a GPCR is attached to a solid phase substrate.

Certain screening methods involve screening for a compound that modulates the expression of the GPCRs described herein, or the levels of natural ligands, e.g., ASP and stanniocalcins. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing the GPCR or ligand and then detecting an increase or decrease in expression (either transcript or translation product). Such assays are typically performed with cells that express the endogenous GPCR or ligand.

Expression can be detected in a number of different ways. As described herein, the expression levels of the protein in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of the GPCR or protein ligand. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques (see above). Alternatively, protein can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to the protein.

Other cell-based assays are reporter assays conducted with cells that do not express the protein. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a promoter that is operably linked to a reporter gene that encodes a detectable product. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, beta-glucuronidase, CAT (chloramphenicol acetyl transferase), luciferase, beta-galactosidase and alkaline phosphatase.

In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either modulates the activity of the promoter by binding to it or triggers a cascade that produces a molecule that modulates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of the GPCR or ligand and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.

In one embodiment the invention provides soluble assays using molecules such as a domain, e.g., a ligand binding domain, an extracellular domain, a transmembrane domain (e.g., one comprising seven transmembrane regions and cytosolic loops), the transmembrane domain and a cytoplasmic domain, an active site, a subunit association region, etc.; a domain that is covalently linked to a heterologous protein to create a chimeric molecule; a GPCR; or a cell or tissue expressing a GPCR, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the domain, chimeric molecule, GPCR, or cell or tissue expressing a GPCR is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microliter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microliter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about. 100-1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention.

The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the signal transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and are appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

Modulators

Inhibitors and/or activators identified according to the methods of the invention can be provided from libraries of compounds available from a number of sources or can be derived by combinatorial chemistry approaches known in the art. Such libraries include but are not limited to the available Chemical Director, Maybridge, and natural product collections. In one embodiment of the invention libraries of compounds with known or predicted structures can be docked to the human β2AR structures of the invention. In another embodiment, the libraries for ligands binding to the ligand binding site can include carazolol and related compounds. In another embodiment, the libraries can include a linker component or moiety. In some embodiments, the linker can include from about 10-22 atoms and can include one or more of C, O, N, S, and/or H atoms. In another embodiment, the libraries can include a ligand binding site (also known as the ligand, agonist, or antagonist binding pocket) component or moiety. In some embodiments, the libraries can include drug-like molecules, i.e., molecules having structural attributes of one or more compounds known to bind to and/or affect a physiologic function of a GPCR.

In some embodiments, the invention includes compounds that can be tested as modulators of GPCR activity. Compounds tested as modulators of GPCRs can be any small chemical compound or biological entity. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps. The assays are typically run in parallel (e.g., in microliter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or ligand libraries are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Russell & Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

It is noted that modulators that compete with the binding and/or activity of the known ligands for to human β2AR can be used to treat various diseases including, but not limited to, coronary artery disease, atherosclerosis, thrombosis, obesity, diabetes, stroke, and other diseases.

In one embodiment, a modulator binds to a site on a GPCR, e.g., a human β2AR. In one aspect, the site is a carazolol binding site. In a related aspect, the site is a ligand binding site. In another aspect, the modulator has a first moiety that binds to a binding site. In another aspect, the first moiety is connected to a linker. In another aspect, the first moiety and the linker are connected to at least one additional moiety that binds to a site other than that bound by the first moiety. In another aspect, the two or more moieties are not connected by a linker and are both present in a composition.

Computer-Based Modeling of β2AR

Protein-ligand docking aims to employ principles by which protein receptors, e.g., human β2AR, recognize, interact, and associate with molecular substrates and compounds to predict the structure arising from the association between a given compound and a target protein of known three-dimensional structure.

In protein-ligand docking, the search algorithm can allow the degrees of freedom of the protein-ligand system to be sampled sufficiently as to include the true binding modes. Three general categories of algorithms have been developed to address this problem of ligand flexibility: systematic methods; random or stochastic methods; and simulation methods.

Systematic search algorithms attempt to explore all degrees of freedom in a molecule. These algorithms can be further divided into three types: conformational search methods, fragmentation methods, and database methods.

In conformational search methods, all rotatable bonds in the ligand are systematically rotated through 360° using a fixed increment, until all possible combinations have been generated and evaluated. As the number of structures generated increases immensely with the number of rotatable bonds (combinatorial explosion), the application of this type of method, in its purest form, is very limited.

Fragmentation methods use two different approaches to incrementally grow the ligands into the active site. One approach is by docking the several fragments into a site and linking them covalently to recreate the initial ligand (“the place-and-join approach”). Another approach is by dividing the ligand into a rigid core-fragment that is docked in first place and flexible regions that are subsequently and successively added (“the incremental approach”). DOCK (see above) is an example of s docking programs that use a fragmentation search method.

Database methods using libraries of pre-generated conformations or conformational ensembles to address the combinatorial explosion problem. A example of a docking program using database methods is FLOG which generates a small set of 25 database conformations per molecule based on distance geometry, that are subsequently subject to a rigid docking protocol.

Random search algorithms sample the conformational space by performing random changes to a single ligand or a population of ligands. At each step, the alteration performed is accepted or rejected based on a predefined probability function. There are three basic types of methods based on random algorithms: Monte Carlo methods (MC), Genetic Algorithm methods (GA), and Tabu Search methods.

Simulation methods employ a rather different approach to the docking problem, based on the calculation of the solutions to Newton's equations of motion. Two major types exist: molecular dynamics (MD) and pure energy minimization methods.

Scoring functions normally employed in protein-ligand docking are generally able to predict binding free energies within 7-10 kJ/mol and can be divided into three major classes: force field-based, empirical, and knowledge-based scoring functions.

In force-field based scoring, standard force fields quantify the sum of two energies: the interaction energy between the receptor and the ligand, and the internal energy of the ligand. The energies are normally accounted through a combination of a van der Waals with an electrostatic energy terms. A Lennard-Jones potential is used to describe the van der Waals energy term, whereas the electrostatic term is given by a Coulombic formulation with a distance-dependent dielectric function that lessens the contribution from charge-charge interactions.

Empirical scoring functions are based on the idea that binding energies can be approximated by a sum of several individual uncorrelated terms. Experimentally determined binding energies and sometimes a training set of experimentally resolved receptor-ligand complexes are used to determine the coefficients for the various terms by means of a regression analysis.

Knowledge-based scoring functions focus on following the rules and general principles statistically derived that aim to reproduce experimentally determined structures, instead of binding energies, trying to implicitly capture binding effects that are difficult to model explicitly. Typically, these methods use very simple atomic interactions-pair potentials, allowing large compound databases to be efficiently screened. These potentials are based on the frequency of occurrence of different atom-atom pair contacts and other typical interactions in large datasets of protein-ligand complexes of known structure. Therefore, their derivation is dependent on the information available in limited sets of structures.

Consensus Scoring combines the information obtained from different scores to compensate for errors from individual scoring functions, therefore improving the probability of finding the correct solution. Several studies have demonstrated the success of consensus scoring methods in relation to the use of individual functions schemes.

Using the Protein-ligand docking methods described above, a predicted association can be made between a selected chemical library compound (see above for examples) and the binding sites in the human β2AR structure described in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). These methods will therefore allow the generation of a binding profile for any known compound in any of the binding sites or cavities of the human β2AR based on the simulated docking of the compound.

In another embodiment, a form of computer-assisted drug design is employed in which a computer system is used to generate a three-dimensional structure of the candidate class A GPCR based on the structural information encoded by the amino acid sequence. This will allow use of the methods described above to identify candidate compounds based on their ability to dock in one or more of the predicted GPCR structure binding sites. In one aspect, the input amino acid sequence of the GPCR interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the class A GPCR. The models of the class A GPCR structure are then examined to identify the position and structure of the binding sites, e.g., a binding pocket. The position and structure of the predicted binding site(s) is then used to identify various compounds that modulate ligand-receptor binding using the methods described above.

The three-dimensional structural model of the GPCR is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a GPCR polypeptide into the computer system. The amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein. At least 10 residues of the amino acid sequence (or a nucleotide sequence encoding 10 amino acids) are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM. The three-dimensional structural model of the GPCR is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art. Any method of protein structure modeling such as ab-initio modeling, threading or sequence-sequence based methods of fold recognition. In one embodiment, the AS2TS system of protein structure modeling is used. In other embodiments, a sequence alignment in combination with a threshold protein sequence similarity to determine a set of protein sequences for which to model protein structure is used. In one aspect, sequence alignments are generated for the set of sequences to be modeled with sequences of proteins with solved empirical structure in a protein structure databank known to one of skill in the art. If the sequences to be modeled have a sufficient similarity to one or more sequences with known protein structure, then the three dimensional structure of the sequence can be modeled.

The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the GPCR of interest. In one embodiment, software can look at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as “energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.

The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.

In another embodiment, protein structure alignments can be used to determine the structure of GPCRs using the known structure of the β2AR (Appendix I) (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance). Protein structure alignments preferably are sets of correspondences between spatial co-ordinates of sets of carbon alpha atoms which form the ‘backbone’ of the three-dimensional structure of polypeptides, although alignments of other backbone or side chain atoms also can be envisioned. These correspondences are generated by computationally aligning or superimposing two sets of atoms order to minimize distance between the two sets of carbon alpha atoms. The root mean square deviation (RMSD) of all the corresponding carbon alpha atoms in the backbone is commonly used as a quantitative measure of the quality of alignment. Another quantitative measure of alignment is the number of equivalent or structurally aligned residues.

In another embodiment, a GPCR structure is calculated based on the solved structure of the human β2AR by computationally aligning or superimposing two sets of atoms to minimize distance between the two sets of carbon alpha atoms (i.e., the alpha carbon atoms of the human β2AR and an unknown GPCR structure), followed by one or more of simulated annealing and energy minimization. The result of this calculation is a computed structure for a GPCR that provides atomic co-ordinates for the alpha carbon backbone as well as side chain atoms.

A variety of methods for generating an optimal set of correspondences can be used in the present invention. Some methods use the calculation of distance matrices to generate an optimal alignment. Other methods maximize the number of equivalent residues while RMSD is kept close to a constant value.

In the calculation of correspondences, various cutoff values can be specified to increase or decrease the stringency of the alignment. These cutoffs can be specified using distance in Angstroms. Depending on the level of stringency employed in the present invention, the distance cutoff used is less than 10 Angstroms or less than 5 Angstroms, or less than 4 Angstroms, or less than 3 Angstroms. One of ordinary skill will recognize that the utility of stringency criterion depends on the resolution of the structure determination.

In another embodiment of the present invention, the set of residue-residue correspondences are created using a local-global alignment (LGA), as described in US Patent Publication Number 2004/0185486. In this method, a set of local superpositions are created in order to detect regions which are most similar. The LGA scoring function has two components, LCS (longest continuous segments) and GDT (global distance test), established for the detection of regions of local and global structure similarities between proteins. In comparing two protein structures, the LCS procedure is able to localize and superimpose the longest segments of residues that can fit under a selected RMSD cutoff. The GDT algorithm is designed to complement evaluations made with LCS searching for the largest (not necessary continuous) set of ‘equivalent’ residues that deviate by no more than a specified distance cutoff.

Using the protein structure alignments described above, the structure of human β2AR in Appendix I (SEQ ID NOS 4-5, 1 and 6-9, respectively in order of appearance) can be used as a model on which to discern the structure of other GPCRs and/or their predicted ligand-binding sites.

Once the GPCR structure has been generated, a binding pocket can be identified by the computer system. Computational models seek to identify the regions by characterization of the three dimensional structure of the GPCR. Some methods of identifying a binding pocket use triangulation such as weighted Delaunay triangulation to determine pocket volumes (castP). Other methods use spheres to determining protein pocket volumes (Q-site-finder, UniquePocket). Conserved binding-site identification seeks to identify conserved regions such as a binding pocket through associating the residues which form the aforementioned regions with conserved residues in homologous protein sequences or structures, e.g., through the use of sequence alignments.

One method of identifying a binding pocket in a GPCR entails filling the three dimensional protein structures with spheres, creating a “negative image” of the structure. A cutoff distance, such as 8 Angstroms, is used to determine spheres which interact with residues. Spheres are labeled as conserved or not-conserved based on their interaction with residues which form a conserved binding site. The conserved spheres are clustered based on their three dimensional co-ordinates to identify a set of spheres with interact with conserved residues and are proximal in three dimensional space forming a cluster. Three-dimensional structures for potential compounds are generated by entering chemical formulas of compounds. The three-dimensional structure of the potential compound is then compared to that of the GPCR protein ligand-binding site(s) (e.g., a binding pocket) to identify compounds that bind to the GPCR binding site(s). Binding affinity between the GPCR binding site(s) and the compound is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.

It should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and can not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the claims.

Reference will now be made in detail to particularly preferred embodiments of the invention. Examples of the preferred embodiments are illustrated in the following Examples section.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed. (Plenum Press) Vols. A and B (1992).

Example 1 Crystallization of a β2AR Protein using a Lipidic Cubic Phase/Sterol Method

This Example describes the generation of diffraction-quality crystals of a PAR protein, specifically crystals of β2AR-T4L, a fusion protein of human β2AR with T4 lysozyme, bound to carazolol. A detailed description of the protein and its synthesis is provided in Example 4. Briefly, T4 lysozyme was generated by three distinct modifications to β2AR: (1) a fusion protein was created by replacement of the third intracellular loop with T4L, (2) the carboxyl terminal 48 amino acids were deleted, and (3) a glycosylation site at Asn187 was eliminated through a glutamate substitution. β2AR-T4L was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, and purified by sequential antibody and ligand affinity chromatography.

LCP Crystallization Setup

Protein solution (30 mg/ml) was mixed with a host lipid or lipid mixture typically in 2:3 ratio by volume ratio using a syringe mixer (Cheng, et al., Chem Phys Lipids 95, 11 (1998). Upon mixing (−100 passages, 2-3 min) the sample spontaneously formed homogeneous transparent cubic phase, which was directly used in crystallization trials. Robotic trials were performed using an in meso crystallization robot (Cherezov, et al., Acta Cryst D 60, 1795 (2004)). Six-well glass sandwich plates (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov, J Appl Cryst 36, 1372 (2003)), were filled with 25 or 50 mL protein-laden lipidic cubic phase drops overlaid by 800 μL of precipitant solution in each well and sealed with a glass coverslip. Manual setups were performed in Impact microbatch plates (Hampton Research cat# HR3-293), Innovaplate SD-2 sitting drop plates (Hampton Research cat# HR3-083) or VDX48 hanging drop plates (Hampton Research cat# HR3-275). Modified repetitive syringe dispenser (Cherezov, et al., J Appl Cryst 38, 398 (2005)), coupled with a gas-tight 100 μL syringe was used to deliver 70 mL of cubic phase drops per well to which 1-2 μL of precipitant solution was added with a microvolume pipette. Reservoirs of the Innovaplate and VDX48 plates were filed with 50 and 100 μL of precipitant respectively. All operations starting from mixing lipid and protein were performed at room temperature (−21-23° C.). After setup, plates were transferred into an automatic incubator/imager (RockImager 1000, Formulatrix Inc.) maintained at 20° C. Plates were imaged every 12 hours for the first 3 days, then every day until 7 days and after that on the 10th and on the 14th day.

Initial Hits

Initial trials were performed using protein solution at a concentration of 30 mg/mL mixed with monoolein as a host lipid against a set of 6 commercial screens (Index HT, SaltRx HT and MemFac HT from Hampton Research, JCSG+ and MbClass from Nextal, and MemSys&MemStart from Molecular Dimensions) set up in duplicates. Initial hits detected in three different wells contained extremely small, <5 μm, needle-like birefringent crystal showers. The detection of such small colorless protein crystals in LCP was made feasible by specially developed glass sandwich plates with optimized optical properties (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov et al., J Appl Cryst 36, 1372 (2003)). Hit conditions were similar by chemical composition containing 30% v/v PEG 400 as a precipitant, low concentration of Li sulfate and a buffer at pH 6.5 or 7.5.

Optimization

Crystal condition optimization is well known to practitioners in the art of x-ray crystallography. What follows is a specific example of a generally applicable optimization approach in which one or more of the crystallization mixture components is systematically varied in concentration or substituted by a chemical analog. Initial rounds of optimization were focused on varying concentration of the main precipitant, PEG 400, buffer pH and identity, and salt identity and concentration. As a result, Li sulfate was replaced with Na sulfate and useful concentration and pH ranges were established. Crystals were still rather small reaching ˜15×5×1 μm in size.

Further, lipid and soluble additives were searched for and optimized simultaneously. Three different host lipids (monopalmitolein, monovaccenin and monoolein), five lipid additives to monoolein host (DOPE, DOPE-Me, DOPC, Asolectin and cholesterol) and 96-well soluble additives screen were tried in different combinations along with the previously found basic crystallization conditions. One of the soluble additives, 1,4-butanediol, stood out, but only when it was used in combination with lipid additives, DOPE or cholesterol. When DOPE was used as an additive the crystals grew as thin plates (40×7×2 μm), while when cholesterol was used the crystals grew as small rods (30×5×5 μm). DOPE was dropped out in favor of cholesterol in the subsequent optimization steps.

Final optimization required fine tuning concentrations of all components (protein, PEG 400, Na sulfate, 1,4-butanediol, cholesterol, buffer pH). At the final stages of optimization, higher concentrations of protein, e.g., 50 mg/mL protein solution, were preferred. Decreasing the volume of lipidic cubic phase per trial from 50 to 20 mL consistently produced larger crystals. The best crystals (40×20×5 μm; FIG. 1) were obtained in 30-35% v/v PEG400, 0.1-0.2 M Na sulfate, 0.1 M Bis-tris propane pH 6.5-7.0, 5-7% v/v 1,4-butanediol using 8-10% w/w cholesterol in monoolein as the host lipid. Thus, in another aspect, the invention provides a method of directly adding a lipid additive (e.g., cholesterol, DOPE) to the host lipid prior to combining with the protein mixture, thereby significantly improving the size and quality of LCP grown crystals. Previously, exogenous lipid had been added only to the protein solution prior to combining the protein solution with the host lipid (Luecke, et al., Science 293, 1499 (2001)).

To limit the range of precipitant and additives concentrations used for screening, previously published phased diagrams were used, e.g., for monoolein (Qiu, et al., Biomaterials 21, 223 (2000)), and monovaccenin (Qiu, et al., J. Phys. Chem. B 102, 4819 (1998)), and the effects on monoolein phase behavior of soluble (Cherezov, et al., Biophys J 81, 225 (2001)), and lipid additives (Cherezov, et al., Biophys J 83, 3393 (2002)). Certain lipid mixtures required pre-screening their phase behavior by microscope visualization and by X-ray, prior to being mixed with the protein for crystallization trials. Thirty-three 96-well optimization screens were used in combinations with different lipid mixtures, resulting in over 15,000 trials. This throughput was made feasible through the use of an in meso crystallization robot and automatic imager.

Typically, the best crystals grown under lipidic cubic phase conditions appear at the boundary between the cubic and the sponge phase (Cherezov, et al., J Mol Biol 357, 1605 (2006); Wadsten, et al., J Mol Biol 364, 44 (2006)). When crystals are close to the phase boundary, equally good crystals are obtained in either phase.

Crystal Harvesting

Crystals were harvested directly from the glass sandwich plates (FIG. 2) because this method provided results superior to those obtained with microbatch or vapor diffusion plates. These plates have been specifically designed to perform optimally only at the screening and optimization stages (Cherezov, et al., Acta Cryst D 60, 1795 (2004); Cherezov and M. Caffrey, J Appl Cryst 36, 1372 (2003)). No harvesting has been previously attempted from them, due to the difficulties in separating glass slides strongly bound by a high performance double sticky tape. Thus, in another aspect, the invention disclosed herein provides a special technique for opening individual wells and harvesting crystals from them. A corner of a capillary cutting stone (in this instance, from Hampton Research) was used to scratch the top glass around the perimeter of the well. Gently pressing the glass slide just outside the scratch allows propagation of the scratch through the depth of the glass. The glass slide was then broken in one point just outside of the scratched perimeter using a sharp needle. This hole was used to lift up the glass slide and expose the cubic phase for harvesting. An extra drop of ˜5 μL of precipitant was added to the well to reduce dehydration. Using this technique, it was possible to open up and harvest crystals successfully from more than 80% of attempted wells.

Crystals were scooped directly from the lipidic cubic phase using 30 μm aperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care was taken to drag as little as possible lipid around the crystal to decrease the unwanted background. Attempts to dissolve the lipids, either by increasing concentration of PEG400 or using a mineral oil, typically resulted in decreasing the diffraction power of the crystals.

Data Collection

During screening for diffraction of these crystals at APS beamline GM/CA CAT (FIG. 3), the crystals themselves could not be observed in the mounted loops (FIG. 1 a). Therefore, a systematic screening of the loop material with varying beams was conducted to identify the crystal in the loop. Optimization of the diffraction with a low x-ray dose was then used to center the crystals and eventually allow for complete data collection using the 10 μm×10 um minibeam setup at GM/CA CAT. The complete data set is then compared to data filtered by a sigma cutoff (see Table 1). All of the data was used in structure solution and refinement.

TABLE 1 # Observed # 

nique Resolution refl refl Redundancy Completeness R_(sym) I/SIGMA R-meas Rmrgd-F Signal/noise −3 10 3352 333 10.1 87.40% 6.60% 23.25 6.90% 2.70% 8 3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80% 6 10480 1003 10.4 99.60% 9.90% 20.01 10.50% 4.00% 3 126008 11968 10.5 99.80% 13.30% 14.01 14.00% 6.60% 2.8 33158 3130 10.6 100.10% 38.00% 6.42 39.90% 18.70% 2.7 19702 1893 10.4 99.70% 49.50% 4.96 52.10% 24.90% 2.6 23772 2275 10.4 99.90% 60.20% 4.07 63.40% 30.10% 2.5 14108 2558 5.5 99.30% 58.90% 2.69 65.10% 51.80% 2.4 14672 3060 4.8 99.10% 67.80% 2.18 75.70% 62.80% total 248843 26574 9.4 99.50% 12.70% 9.62 13.40% 11.40% Signal/noise 0 10 3352 333 10.1 87.40% 6.60% 23.25 6.90% 2.70% 8 3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80% 6 10480 1003 10.4 99.60% 9.90% 20.01 10.50% 4.00% 3 125628 11923 10.5 99.40% 13.30% 14.06 14.00% 6.50% 2.8 32679 3077 10.6 98.40% 37.60% 6.54 39.50% 17.70% 2.7 19346 1849 10.5 97.40% 48.80% 5.08 51.30% 23.40% 2.6 23201 2210 10.5 97.00% 58.90% 4.2 62.00% 28.00% 2.5 13461 2406 5.6 93.40% 56.40% 2.88 62.30% 45.90% 2.4 13833 2827 4.9 91.50% 64.10% 2.4 71.50% 54.30% total 245571 25982 9.5 97.30% 12.60% 9.85 13.30% 10.80%

indicates data missing or illegible when filed

Example 2 Using the LCP/Sterol Method to Generate Additional Membrane Protein Crystals

In addition to the β₂AR-T4L/carazolol structure (Examples 1, 3, and 4), the LCP/sterol matrix has successfully been used to crystallize a diversity of receptor-ligand systems.

1. β₂AR-T4L^((E122W)) A thermally-stabilized construct of β₂AR-T4L comprising an E122W mutation has been crystallized in the presence of both agonist and antagonist ligands including: alprenolol, timolol, clenbuterol and carazolol. For lipidic cubic phase (LCP) crystallization of β₂AR-T4L^((E122W)), robotic trials were performed using an in meso crystallization robot (Cherezov et al., 2004). Glass sandwich plates in 96-well format (Cherezov and Caffrey, 2003; Cherezov et al., 2004) were filled with 25 or 50 mL protein-laden LCP drops overlaid by 0.8 μL of precipitant solution in each well and sealed with a glass coverslip. All operations starting from mixing lipid and protein were performed at room temperature (−21-23° C.). Crystals were obtained in 28% (v/v) PEG 400, 0.3 M potassium formate, 0.1 M Bis-tris propane pH 7.0 and saturating concentrations of ligand (e.g., 2 mM in the case of timolol) using 10% (w/w) cholesterol in monoolein as the host lipid. Diffraction data were collected on all four ligand complexes (see FIG. 4), and structures were determined for alprenolol (3.2 Å), timolol (2.8 Å), and carazolol (2.8 Å).

2. A_(2A)R-T4L The applicability of the monoolein cholesterol system in the crystallization of non-biogenic amine receptors has also been demonstrated with the structural determination of the human A_(2A) adenosine receptor (A_(2A)R-T4L) bound to a high affinity selective antagonist, ZM241385, to 2.6 Å resolution. See FIG. 4. For lipidic cubic phase (LCP) crystallization of the human A2A adenosine receptor in meso, glass sandwich plates (Cherezov, et al., Acta Crystallogr D Biol Crystallogr, 60, 1795 (2004)) were filled with 50 n1 receptor-cholesterol-monoolein LCP drops overlaid by 0.8 μl of precipitant solution in each well and sealed with a glass coverslip. Lipid:receptor LCP mixture typically contained monoolein:cholesterol (54%:6% (w/w)) and receptor (40% (w/w)). Crystallization set-ups were performed at ambient temperature (22±2° C.). Plates were incubated and imaged at 20° C. using an automated incubator/imager (RockImager 1000, Formulatrix). Data-collection quality crystals (−100 μm×10 μm×5 μm) were obtained in 30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate (range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5 to 6.5) and 200 μM ZM241385. The protein crystallized in the primitive monoclinic space group P2₁ with one molecule per asymmetric unit and an estimated solvent content of 52%.

3. β₂AR^((E122W)) Initial crystals of β₂AR^((E122W)), lacking inserted T4 lysozyme, have also been obtained. The protein was extracted from insect cell membranes using a mixture of 0.5% w/v dodecyl maltoside (DDM), 0.1% w/v cholesteryl hemisuccinate (CHS) and 1 mM timolol. Timolol was maintained at 1 mM throughout the first steps of the purification. The extracted protein was purified by binding overnight to Talon immobilized metal affinity resin followed by a standard washing and elution with 200 mM imidazole. Adenosine triphosphate at 5 mM in combination with 10 mM MgCl₂ was used to eliminate chaperone protein contamination. Eluted protein was concentrated to 2.5 mL and desalted into a 0 mM imidazole buffer using a PD10 desalting column (GE-Biosciences). Protein was then bound to 1004 of Ni-sepharose immobilized metal affinity resin in the presence of PNGase (New England Biolabs) to remove glycosylation, and incubated overnight. After incubation the protein was washed on the column and timolol was replaced by carazolol for structure solution. The protein bound to carazolol was eluted from the Ni-Sepharose column, treated with 100 mM Nacitrate pH 7.5, and concentrated to 50 mg/mL.

The protein solution was then mixed with monoolein containing 10% cholesterol at a ration of 40:60% w/w protein to lipid to generate the lipidic cubic phase used in crystallization trials. The LCP lipid containing protein was dispensed onto glass sandwich crystallization plates at a volume of 20 mL to which 1 μL of crystallization solution was added. The entire experiment in 96 well format was then covered by an additional glass plate which was fastened to the first by virtue of double back sticky tape. Initial crystals have been obtained after 24 hours by addition of a solution containing 35% v/v PEG 400, 100 mM NaSO4 100 mM Bis tris propane pH 7 and 8% 2,6 hexanediol.

By way of a prophetic example, optimized crystals of β₂AR^((E122W)) obtained by this method are screened for their ability to diffract at high resolution, e.g., less than 3.5 Å or, more preferably, less than 3 Å. Guidance for optimization is provided by the optimization protocols set forth herein and in the examples. In combination with the teaching provided herein, one skilled in the art will readily identify appropriate beam settings to obtain diffraction patterns from which a detailed molecular structure of the optimally crystallized protein can be solved.

4. CXCR4-T4L CXCR4, also called fusin, is a GPCR protein specific for stromal-derived-factor-1 (SDF-1 also called CXCL12), a molecule endowed with potent chemotactic activity for lymphocytes. This Example teaches prophetically how the methods of the invention may be used to generate diffraction quality crystals of a fusion protein comprising CXCR4 (CXCR4-T4L).

The cDNA encoding CXCR4 is synthesized by outsourcing to DNA2.0 where the DNA was optimized for human codon usage, elimination of transcribed RNA secondary structure, elimination of ribosome binding sites and avoidance of common restrictions sites used in subsequent cloning procedures. Two initial variants are contracted to be synthesized, the first encoding a wild-type full-length receptor and the second a full length receptor with a fusion protein located between transmembrane helix V and helix VI, effectively eliminating the third intracellular loop (3IL) region of the receptor. In the case of CXCR4, T4-lysozyme (T4L) is the fusion protein fused in the place of the 3IL. A set of guidelines is followed for the incorporation of T4L into the fusion protein which minimizes the possibility of structural disruption and concomitant effects on protein expression and stability. Briefly, the 5′ insertion point for the fusion protein takes precedence over the 3′ insertion point and is located 66 nucleotides (22 residues) downstream of the codon for a conserved proline on helix V of the receptor. If the 3IL section of the receptor is large, the 3′ fusion point is set 87 nucleotides (29 residues) upstream from the codon for the family conserved proline on helix VI. However, as is the case for CXCR4, where the 3IL loop is small, cDNA for the fusion protein is inserted directly into the 3IL loop position dictated by spacing from helix V with no resulting excision of intervening nucleotides. Specifically, T4L is inserted into CXCR4 based on spacing between a conserved proline on helix V. and a C-terminal truncation was generated based on literature precedence. Each synthesized cDNA is flanked by an out of frame AscI (GGCGCGCCG) restriction site on the 5′ end and an in frame FseI (GGCCGGCC) on the 3′ end for sub-cloning into a set of four expression vectors. Viral DNA is then generated, amplified from these vectors according to standard protocols and titered using flow cytometry to measure the population of cells expressing the virally encoded GP64 protein.

Protein Expression of CXCR4-T4L

With titered virus in hand, small scale expression trials are carried out in a volume of 5 mL/experiment. Expression levels are assessed using flow cytometry to measure the mean fluorescence intensity (MFI) and percentage of cells expressing the FLAG epitope encoded by the expression screening vectors. Expressing cells are tested with and without permeabilization to generate a ratio between protein inserted in the plasma membrane and protein inside the cellular trafficking machinery. A correlation between cell surface expression and overall protein expression is demonstrated, as well as a correlation between stability and the ratio of cell surface expression/total expression. In addition to these assays, small scale purification after solubilization with dodecyl maltoside (DDM) is carried out to determine the quantity of recoverable protein as well as the quality as measured by size exclusion chromatography. Based on these data it was apparent that the T4L fused receptor is expressing and that it is dimerizing in a ligand independent manner, an indicator of C-terminal non-specific interactions in other receptors. Thus, a C-terminal truncation mutant of CXCR4 is generated.

Protein Purification

The C-terminal truncation of T4L fused CXCR4 was scaled up to production sized expression (5-10 L of cell culture) and further processed by large scale purification efforts intended for crystallization trials. Briefly 5-10 L of cells culture are centrifuged and washed with PBS followed by freezing at −80° C. The frozen cellular material is then resuspended in 820 mL of lysis buffer (10 mM Hepes pH 7.5, 10 mM MgCl₂, 20 mM KCl) supplemented with protease inhibitor (Roche). The cell suspension is lysed by 20 strokes of a dounce homogenizer and centrifuged at 45,000 rpm in Ti45 ultracentrifuge for 30 minutes. The resulted pellet was separated from the supernatant, resuspended and the process repeated six times to ensure complete removal of soluble protein material. On the final resuspension step the membranes were resuspended in 100 mL of lysis buffer containing 40% v/v glycerol, homogenized with 20 strokes of a dounce homogenizer and frozen in 10 mL aliquots at −80° C. for indefinite storage.

For solubilization and purification, each 10 mL aliquot of frozen membranes is resuspended to 25 mL using lysis buffer to which 100 uM AMD070 ligand is added in addition to protease inhibitor at 2× working concentration and 2 mg/mL iodoacetamide. The membranes are allowed to thaw and incubate with ligand at an appropriate temperature for at least 30 minutes. After the incubation the mixture is diluted two-fold with a 2× solubilization buffer containing 100 mM Hepes pH 7.5, 1M NaCl, 2% w/v DDM 0.2% w/v CHS. The solubilization is allowed to proceed with agitation for at least 2 hours at 4° C. after which insoluble material is separated by centrifugation and discarded. The supernatant is isolated and allowed to bind to 0.5 mL of Talon (Clontech) IMAC resin charged with Co²⁺ in the presence of 20 mM imidizole buffered to 7.5 and 800 mM NaCl. Binding to the Talon IMAC resin is allowed to proceed with agitation at least 4 hours but most commonly overnight. After binding, the slurry is poured into a gravity column and the resin is separated from the supernatant. The resin is then washed with 80 column volumes (CV) of wash buffer (50 mM Hepes pH 7.5, 800 mM NaCl, 20 mM Imidizole, 0.1% w/v DDM, 0.01% w/v CHS and 100 uM AMD070 (or receptor appropriate ligand). After the initial wash the resin is further treated to adjust the NaCl concentration to 500 mM and to increase the ligand concentration to 300 uM. The protein is then eluted from the resin using 200 mM Imidazole and concentrated to 2.5 mL for removal of the excess imidizole with a PD10 desalting column (GE Biosciences). The ligand concentration is increased to 500 uM and the protein is bound to 100 uL of Ni-Sepharose IMAC resin in the presence of 20,000 units of PNGase (NEB) an endoglycosidase capable of removing N-linked glycosylation. The protein is allowed to bind to the resin and deglycosylate for 6 hours after which the resin is washed with imidizole free elution buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 0.05% w/v DDM, 0.01% w/v CHS and 1 mM AMD 070). After the washing step the protein was eluted from the resin using the same buffer but including 200 mM imidizole. After elution the protein is normally concentrated to approximately 50 mg/mL and tested for integrity by SEC. Crystallizable protein should be >90% free of heterogeneity as judged by SDS-PAGE and contain no detectable aggregated species at high protein concentrations as judged by SEC. If the protein remains of high quality it is reconstituted into lipidic cubic phase containing cholesterol. The reconstituted protein is then dispensed onto glass sandwich crystallization plates and tested for crystallization using the screening methodology described in this Example and Example 1. After mixing, the protein-laden lipid cubic phase mixture will comprise 3.6-7.2% w/w sterol, 56.5-52.8 w/w % Monoolein and 40% w/w protein solution (a 3:2 ratio of lipid mixture to protein). Initial crystallization conditions use PEG 400 between 25-35%, a salt between 50-500 mM, and a pH between 5.0-7.5.

Example 3 High Resolution Crystal Structure of a Human 11-Adrenergic G protein-Coupled Receptor T4 Lysozyme Fusion Protein

The engineering, functional properties, expression and purification of crystallization grade β2AR-T4L protein are described in more detail in Example 4. Briefly, β2AR-T4L was generated by three distinct modifications to β2AR: (1) a fusion protein was created by replacement of the third intracellular loop with T4L, (2) the carboxyl terminal 48 amino acids were deleted, and (3) a glycosylation site at Asn187 was eliminated through a glutamate substitution. β2AR-T4L was expressed in Sf9 insect cells, solubilized in 1% dodecylmaltoside, and purified by sequential antibody and ligand affinity chromatography. Using the modified lipidic cubic phase (LCP) crystallization procedure described herein, wherein crystals are grown from a cholesterol-doped monoolein cubic phase, β2AR-T4L crystals were obtained that diffract to a resolution of 2.2 Å. The structure was solved at 2.4 Å resolution. Compared to crystallization in detergents, LCP provides a more native, lipid environment for crystallization, as well as a confinement of protein molecules to two-dimensional membrane sheets that may facilitate the crystallization process through the formation of Type I packing interactions (Caffrey, Curr Opin Struct Biol 10, 486 (2000); Deisenhofer, EMBO J. 8, 2149 (1989); Landau et al., Proc Natl Acad Sci USA 93, 14532 (1996)).

Methods

Lipidic cubic phase crystallization. Crystals of engineered human β2AR (β2AR-T4L) grown from bicelles could not be optimized beyond 3.5 Å resolution (FIG. 5). Lipidic cubic phase (LCP) crystallization trials were therefore performed using an in meso crystallization robot (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004)). 96-well glass sandwich plates (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J Membr Biol 195, 165 (2003)) were filled with 25 or 50 mL protein-laden LCP drops overlaid by 0.8 μL of precipitant solution in each well and sealed with a glass coverslip. All operations starting from mixing lipid and protein were performed at room temperature (−21-23° C.). Crystals were obtained in 30-35% (v/v) PEG 400, 0.1-0.2 M sodium sulfate, 0.1 M Bis-tris propane pH 6.5-7.0 and 5-7% (v/v) 1,4-butanediol using 8-10% (w/w) cholesterol in monoolein as the host lipid (FIG. 6A). Addition of cholesterol and 1,4-butanediol dramatically improved crystals size and shape, thereby enabling high-resolution diffraction. In this instance, additions of phospholipids (dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, asolectin) to the main host LCP lipid monoolein (either alone or in combination with cholesterol) failed to improve crystal quality.

Crystal Harvesting

The average size of the harvested crystals was 30×15×5 μm (largest crystal was 40×20×7 μm). Crystals were harvested directly from the glass sandwich plates, even though these plates have been specifically designed for screening and optimization (Cherezov, et al., Acta Crystallogr D Biol Crystallogr 60, 1795 (2004); Cherezov, et al, J Membr Biol 195, 165 (2003)). Crystals were scooped directly from the LCP using 30 or 50 μm aperture MiTeGen MicroMounts and plunged into liquid nitrogen. Care was taken to drag as little as possible lipid around the crystal to decrease unwanted background scattering. Attempts to dissolve the lipids, either by increasing concentration of PEG 400 or using a mineral oil, typically resulted in a decrease in diffraction power of the crystals.

Data Collection

X-ray data were collected on the 231D-B beamline (GM/CA CAT) at the Advanced Photon Source, Argonne, IL using a 10 μm minibeam (wavelength 1.0332 Å) and a MarMosaic 300 CCD detector (FIG. 6B). Several complete datasets were collected from single crystals at resolution between 2.8 and 3.5 Å using 5× attenuated beam, 5 s exposure and 1° oscillation per frame. However, some crystals diffracted to a maximum of 2.2 Å resolution upon 5 s exposure with 1× attenuated beam. Therefore, 10-20° wedges of high-resolution data were collected from more than 40 crystals (some of the crystals were large enough to allow 2-3 translations). 31 of the best datasets from 27 independent crystals were then combined and scaled against the lower resolution full dataset to obtain complete 2.4 Å data.

One of the challenges during data collection was visualization of colorless microcrystals within an opaque frozen lipid phase and aligning them with the 10 μm minibeam. Because the crystals could not be adequately visualized through the inline optics at the beamline, alignment-by-diffraction techniques were employed. The present invention provides, in one aspect, an optimized crystal search algorithm to locate the crystals without the minibeam. First, the area of the loop containing lipid was scanned in the vertical direction with a highly attenuated and slitted 100×25 μm beam. When diffraction was found, the crystal location was further confined by two additional exposures to an area of ˜50×25 μm. This area was further coarse-scanned with the collimated and 10× attenuated minibeam using 15 μm steps, following by fine-tuning the position using 5 and 2 μm steps. After locating the crystal in one orientation the loop was rotated 90° and the procedure was repeated. Typically during alignment the crystal was exposed ˜10 times using 10× attenuated beam and 2 s exposures.

Data Processing

A 90% complete, 2-fold redundant monoclinic dataset was processed from one crystal diffracting to 2.8 Å resolution. Initial indexing of lattice parameters in spacegroup C2 and crystal orientation were performed using HKL2000 (Otwinowski, et al, in Methods in Enzymology C. W. J. Carter, R. M. Sweet, Eds. (Academic Press, New York, 1997), vol. 276, pp. 307-326). The refined lattice parameters and space group were implemented in the data processing program XDS for spot integration which models error explicitly for radiation decay, absorption, and rotation (Kabsch, J Appl Crystallogr 26, 795 (1993)). Because data was collected using a 10 μm beam from microcrystals, maintaining the crystal orientation at the beam center during data collection was especially problematic. It appeared that XDS modeled the crystal orientation error upon rotation about the phi axis better than other data processing programs that were tried, resulting in better merging statistics. In addition to rotational error, the radiation decay was also an issue that was partially corrected by the XDS processing program, enabling a more reliable scaling of datasets from different crystals and translations of crystals. The 2.8 Å data was used as a scaling reference for incorporation of additional wedges of data collected at a much higher exposure. Each new dataset was indexed in XDS using the original unit cell parameters as constants which were then refined along with the crystal orientation, beam geometry, and mosaicity parameters. The refinement was generally stable, resulting in very similar unit cell constants which enabled subsequent scaling. All of the integrated wedges of data were then tested individually against the scaling reference set and included in the final scaled dataset if the merging statistics remained acceptable upon incorporation of the data. In total, 31 wedges of data from 27 crystals were combined with the scaling reference dataset, 22 of which diffracted to a resolution of 2.4 Å or better. Each of the higher resolution datasets were exposed to a much larger dose of radiation resulting in a rapid decay in intensity. Typically 10°-20° wedges were collected from each crystal or translation, 5°-7° of which had diffraction data to 2.4 Å. The final merging statistics for the dataset are shown in Table 2. Based on the mean F/a(F) of reflections near the three crystallographic axes, the effective resolution is estimated to be 2.4 Å along b* and c* and 2.7 Å along a*. The anisotropy results in the high merging R factors in the last few resolution shells despite the significant I/σ(I) values. The anisotropy is either an inherent property of the crystals or the result of a preferential orientation of the crystals within the mounting loop. Thus, the higher resolution shells were filled in anisotropically by incorporation of the additional data at high exposure levels, while the lower resolution shells have a very high redundancy and low anisotropy.

TABLE 2 Data processing statistics from XDS. A comparison is made between data filtered by a sigma cutoff and the complete set. All of the data was used in structure solution and refinement. # Observed # Unique Resolution refl refl Redundancy Completeness R_(sym) I/SIGMA R-meas Rmrgd-F Signal/noise ≧ −3 10 3352 333 10.1 87.40% 6.60% 23.25 6.90% 2.70% 8 3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80% 6 10480 1003 10.4 99.60% 9.90% 20.01 10.50% 4.00% 3 126008 11968 10.5 99.80% 13.30% 14.01 14.00% 6.60% 2.8 33158 3130 10.6 100.10% 38.00% 6.42 39.90% 18.70% 2.7 19702 1893 10.4 99.70% 49.50% 4.96 52.10% 24.90% 2.6 23772 2275 10.4 99.90% 60.20% 4.07 63.40% 30.10% 2.5 14108 2558 5.5 99.30% 58.90% 2.69 65.10% 51.80% 2.4 14672 3060 4.8 99.10% 67.80% 2.18 75.70% 62.80% Total 248843 26574 9.4 99.50% 12.70% 9.62 13.40% 11.40% Signal/noise ≧ 0 10 3352 333 10.1 87.40% 6.60% 23.25 6.90% 2.70% 8 3591 354 10.1 99.40% 7.60% 22.6 8.00% 2.80% 6 10480 1003 10.4 99.60% 9.90% 20.01 10.50% 4.00% 3 125628 11923 10.5 99.40% 13.30% 14.06 14.00% 6.50% 2.8 32679 3077 10.6 98.40% 37.60% 6.54 39.50% 17.70% 2.7 19346 1849 10.5 97.40% 48.80% 5.08 51.30% 23.40% 2.6 23201 2210 10.5 97.00% 58.90% 4.2 62.00% 28.00% 2.5 13461 2406 5.6 93.40% 56.40% 2.88 62.30% 45.90% 2.4 13833 2827 4.9 91.50% 64.10% 2.4 71.50% 54.30% Total 245571 25982 9.5 97.30% 12.60% 9.85 13.30% 10.80% R_(sym) = Σ_(hkl)|I(hkl) − <I(hkl)>|/Σ_(hkl)(hkl), where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl); R-meas = redundancy independent Rsym; Rmrgd-F = indication of amplitude quality. See, e.g., Murshudov, et al, Acta Crystallogr D D53, 240 (1997).

Structure Solution and Refinement

Initial phases for β2AR-T4L were obtained by molecular replacement using both T4-lysozyme (PDB ID Code 2LZM) and a polyalanine model of the rhodopsin seven-transmembrane bundle (PDB ID Code 1U19) as search models. It was necessary to trim the lysozyme search model to remove residues 12-71 as that domain had shifted conformations relative to the larger section. This domain was later reintroduced to the model by fitting into observed density. Molecular replacement was carried out using the program Phaser by first placing the truncated lysozyme (RFZ=3.74; TFZ=3.65) followed by the rhodopsin model (RFZ=5.2; TFZ=7) (McCoy, Acta Crystallogr D Biol Crystallogr 63, 32 (2007)). In order to optimize placement of the receptor each of the seven helices was refined independently by rigid body maximum likelihood refinement as implemented in Refmac (Initial Rwork/Rfree=0.50/0.51) (Murshudov, et al, Acta Crystallogr D D53, 240 (1997)).

Initial rounds of refinement were carried out using restrained parameters in Refmac. Model rebuilding was performed in Coot utilizing 2 Fo-Fc sigma-A weighted maps, as well as density modified maps calculated using Resolve prime-and-switch phasing which reduces model bias introduced by model based phasing methods (Terwilliger, Acta Crystallogr D D56, 965 (2000)). The Resolve calculated maps were superior to the sigma-A weighted ones in that more of the main chain density could be traced. Density for the bound ligand was visible early in the refinement but was not modeled immediately to allow an unbiased assessment of the phase quality through the improvement of the signal/noise of the observed ligand density. The structure quality is excellent (Table 3), with strong electron density in particular observed in the ligand binding site (FIG. 7), cholesterol binding sites (FIG. 8A), and the proline helix kinks (FIG. 8B).

TABLE 3 Data collection and refinement statistics β₂AR-T4L Data collection (APS GM/CA CAT 23ID-B, 10 μm beam)* Space group C2 Cell dimensions a, b, c (Å) 106.32, 169.24, 40.15 β (°) 105.62 No. of reflections processed 245,571 No. unique reflections 26,574 Resolution (Å) 50-2.4 (2.5-2.4) R_(sym) 12.7 (67.8) Mean I/σ (I) 9.6 (2.2) Completeness (%) 99.5 (99.1) Redundancy 9.4 (4.8) Refinement* Resolution (Å) 20-2.4 (2.46-2.4) No. reflections (test set) 25,247 (1,310) R_(work)/R_(free) 19.8 (27.0)/23.2 (30.1) No. atoms 3,805 Protein 3,544 Ions, lipids, ligand and other 213 Water 48 Overall B-values (Å²) 82 β₂AR 77 T4-Lysozyme 75 Carazolol 55 Lipid 100 R.m.s deviations Bond lengths (Å) 0.013 Bond angles (°) 1.5 Ramachandran plot statistics (%) (excl. Gly, Pro): Most favored regions 94.8 Additionally allowed regions 5.0 Generously allowed regions 0.2 Disallowed regions 0 *Highest resolution shell is shown in parenthesis. R_(sym) = Σ_(hkl)|I(hkl) − <I(hkl)>|/Σ_(hkl)(hkl), where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl).

Analysis of β2A Receptor Topology

The final model of β2AR-T4L includes 442 amino acids. β2AR-T4L was treated with iodoacetamide during purification to eliminate free thiols. The model includes a palmitic acid covalently bound to Cys341 (GPCRs are frequently post-translationally modified with palmitoylate on cysteine residues at the C-terminal tail) and an acetamide molecule bound to Cys265^(6.27). Throughout the description, residues are designated by their position within the β2AR sequence and their Ballesteros-Weinstein designation as a superscript where applicable. Ballesteros-Weinstein numbering is used throughout the text as superscripts to the protein numbering. Within each helix is a single most conserved residue among the class A GPCRs. This residue is designated x.50 where x is the number of the transmembrane helix. All other residues on that helix are numbered relative to this conserved position. The model also includes one carazolol molecule, three cholesterol molecules, two sulfate ions and two butanediol molecules that interact with β2AR. There are also four sulfate ions, a putative disaccharide (modeled as maltose) and a molecule of PEG 400 bound to T4L. For PAR, excellent electron density is observed for residues 29-342, including the ligand carazolol and the two disulfide bonds Cys106^(3.25)-Cys191^(5.30) and Cys184^(4.76.)Cys190^(5.29). The palmitic acid at Cys341 is clearly visible in Fo-Fc omit maps; however, the quality of the electron density is lower than for the rest of the receptor. The N-terminus (residues 1 to 28) and the majority of the C-terminus (residues 343 to 365) are disordered and not visible in the structure.

The β2AR has a fold composed of seven transmembrane helices forming a helical bundle (FIG. 9A). The residues that make up the helices (I to VII) in β2AR are as follows: helix 129^(1.28) to 60^(1.59), helix II 67^(2.38) to 96^(2.67), helix IR 103^(3.22) to 136^(3.55), helix IV 147^(4.39) to 171^(4.63), helix V 197^(5.36) to 229^(5.68), helix VI 267^(6.29) to 298^(6.60), and helix VII 305^(7.32) to 328^(7.55). The residues forming the intracellular loops (ICL) and extracellular loops (ECL) of β2AR are: ICL1 61^(1.60) to 66^(2.37), ECL1 97^(2.68) to 102^(3.21), ICL2 137^(3.36) to 146^(4.38), ECL2 172^(4.64) to 196^(5.35), ICL3 230^(5.69) to 266^(6.28) (residues 231 to 262 are replaced by T4-lysozyme residues 2 to 161), and ECL3 299^(6.61) to 304^(7.31). Helices II, V, VI and VII each have a proline-induced kink at conserved positions along the span of the transmembrane segments. These kinks are thought to enable the structural rearrangements required for activation of G protein effectors (Yohannan et al., Proc Natl Acad Sci USA 101, 959 (2004)). In addition to the seven membrane spanning helices, β2AR possesses two other helical segments: helix VIII, which is believed to be common to all rhodopsin-like GPCRs (Katragadda et al., Biochim Biophys Acta 1663, 74 (2004)), and an unexpected, short helical segment in the middle of ECL2, which is not present in rhodopsin, and was not predicted by computational secondary structure analysis (FIG. 9A).

In the β2AR-T4L construct, T4L is fused to the truncated cytoplasmic ends of helices V and VI. In the crystal structure, the T4L moiety is tilted slightly away from the center axis of β2AR drawn normal to the membrane (FIG. 9B). As a result, interactions between T4L and β2AR are minimal, with only 400A2 of surface area buried between them. The intramolecular contacts between T4L and β2AR include salt bridges between the side chains of T4L-Asp159 and the side-chain amine of β2AR-Lys227^(5.66) (distance 3.4 Å) and between the guanidinium group of T4L-Arg8 with the side-chain carboxyl of β2AR-Glu268^(63.0) on helix VI (distance 3.2 Å) (FIG. 9C, Table 4). The latter interaction is noteworthy because it differs from rhodopsin where Glu6.30 forms an ionic bond with Arg3.50 of the conserved D(E)RY motif. This interaction is postulated to be important for maintaining rhodopsin in the inactive state, but the charged groups of the two residues [Arg131^(3.50) (NH1) and Glu268^(63.0) (OE1)] are 10 Å apart in the β2AR-T4L structure. The remainder of the lysozyme molecule provides important crystal packing interactions, but does not appear to influence significantly the receptor structure.

TABLE 4 Direct contacts between β₂AR and T4L β₂AR atom T4 Lysozyme Distance (Å) van der Waals Contacts Leu230^(5.69) Trp 158 Lys263^(6.25) Asp159 Cys265^(6.27) Ile9 Leu266^(6.28) Ile9 Leu266^(6.28) Glu5 Hydrogen Bond and Salt Bridge Contacts Lys227^(5.66) (NZ) Asp159 (OD1) 3.4 Gln229^(5.68) (O) Asn2 (N) 3.1 Gln229^(5.68) (O) Asn2 (ND2) 3.2 Gln268^(6.30) (OE2) Arg8 (NH2) 3.2 Covalent bonds Leu230^(5.69) Asn2 Lys263^(6.25) Tyr161

Crystal Packing Interactions

The β2AR-T4L protein is packed in a C-centered monoclinic lattice with one molecule per asymmetric unit (FIG. 10A). Membrane protein generally can form two types of crystal packing: Type I represents stacks of two dimensional crystals ordered in the third dimension via interactions of hydrophilic parts of membrane proteins. Type II crystals are composed of membrane proteins whose hydrophobic part is shielded by a detergent micelle and all crystal contacts are formed through hydrophilic, solvent exposed parts of protein molecules. As observed in all previous lipidic mesophase grown crystals (Schwartz, et al., Annu Rev Pharmacol Toxicol 46, 481 (2006)), the β2AR-T4L crystals adopt Type I packing, featuring a multilayered arrangement in accordance with proposed crystallization mechanism (Caffrey, Curr Opin Struct Biol 10, 486 (2000); P. Nollert, et al., FEBS Lett 504, 179 (2001)). Within each layer, protein molecules form arrays of parallel, symmetry-related dimers. There are four distinct crystal-packing interactions within each layer, three of which are mediated by T4L. The fourth interaction in the array is between two receptor molecules related by a crystallographic two-fold rotation axis. This is the sole interaction between symmetry-related receptors, and is mediated primarily by ordered lipids consisting of six cholesterol and two palmitic acid molecules, the latter being covalently attached to Cys341 in the C-terminal portion of the receptor (O'Dowd et al., J Biol Chem 264, 7564 (1989)) (FIG. 10B). These eight lipid molecules form a two-fold symmetric sheet between receptors. The only direct receptor-receptor contact involves a 2.7 Å pair of ionic interactions between the charged amine group of Lys60^(1.59) in helix I and the carboxylate of Glu^(3.38) in helix VIII from the symmetry-related receptor. Remarkably, of the 515 Å2 buried at the receptor symmetry interface, 73% of the crystal contact surface area is mediated by ordered lipid, while only 27% is contributed by protein-protein contacts. The stacking interactions between layers are formed between T4L and extracellular loops ECL2 and ECL3 of the receptor (FIG. 10A). It is unlikely that these contacts affect the orientation of these loops due to the small size of ECL3 and the rigid architecture of ECL2.

Lipid Mediated Receptor Association

Many GPCRs including β2AR are thought to exist as dimers in the plasma membrane, although the location of the dimer interface and the functional significance of dimerization is not clear (Milligan, Mol Pharmacol 66, 1 (2004)). The observation of ordered lipids in the helix I and VIII interface between two symmetry related molecules suggests the association is physiologically relevant (Angers, et al., Proc Natl Acad Sci USA 97, 3684 (2000); Javitch, Mol Pharmacol 66, 1077 (2004); Mercier, et al., J Biol Chem 277, 44925 (2002)). Associations between the equivalent regions of rhodopsin have been found in crystal structures (Salom et al., Proc Natl Acad Sci USA 103, 16123 (2006); Schertler, Curr Opin Struct Biol 15, 408 (2005)) (FIG. 10C).

The role of cholesterol in the physiologic function of β2AR is well documented. Depletion of cholesterol from the membranes of neonatal cardiac myocytes alters the signaling behavior of endogenous β2AR (Xiang, et al., J Biol Chem 277, 34280 (2002)). In untreated cells, activation of β2AR results in sequential coupling to the G proteins Gs and Gi, producing a biphasic effect on myocyte contraction rate. Upon depletion of cholesterol, the β2AR couples more strongly to Gs. This effect may be due to a role of cholesterol in regulating interactions between the β2AR and G proteins, or an effect of cholesterol on β2AR dimerization. The β2AR couples efficiently to Gs as a monomer (Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), so cholesterol mediated association (dimerization) may reduce the efficiency of β2AR coupling to Gs. The effects of cholesterol depletion on β2AR signaling may also be a secondary effect of altering subcellular signaling compartments. There is evidence that cells may concentrate signaling molecules, such as GPCRs and their cognate G proteins, by way of membrane microdomains or compartments, such as caveolae (Ostrom, et al., Br J Pharmacol 143, 235 (September, 2004)). This compartmentalization may be a major regulator of receptor-effector coupling. Thus, the importance of cholesterol in forming the observed crystallographic association is consistent with its role in β2AR signaling.

Electrostatic Charge Distribution

Electrostatic charge distribution was calculated using APBS (Gether, Endocr Rev 21, 90 (2000)) and mapped onto a molecular surface representation of β2AR. The analysis reveals three polarized areas within the molecule (FIG. 11A). First, the cytoplasmic face of the receptor is involved in G protein interaction and carries a net positive charge even in the absence of ICL3, which also has a predicted overall positive charge (FIG. 11B). The second site is an electrostatically negative region located within the membrane between helices HI, IV and V potentially exposed to the lipid alkyl chains, which is unexpected as the burial of charge within the plasma membrane is thermodynamically unfavorable. A glutamate residue at position 122^(3.41) may partially account for the observed charge distribution. Finally, the binding site cleft is negatively charged and exposed to solvent by an unusual ECL2 architecture and lack of N-terminal interactions. This negative charge may facilitate ligand binding through electrostatic funneling of positively charged catecholamines (FIG. 11B).

Extracellular Region

The ECLs and amino termini of GPCRs, together with the extracellular halves of the transmembrane helices, are believed to define the ligand-binding site of each receptor (Angers et al., Proc Natl Acad Sci USA 97, 3684 (2000)). Therefore, the ECLs play an important role in the overall pharmacology of any particular receptor. In general, small molecule ligands are thought to bind deeper within the space created by the transmembrane domain helices, whereas larger ligands such as peptides bind closer to the membrane surface near the ECLs (Ji, et al., J Biol Chem 273, 17299 (1998); Gether, Endocr Rev 21, 90 (2000)). Mutagenesis studies suggest that the β2AR binds its ligand deep within the transmembrane helix bundle, which may be related to the observation that the extracellular regions have a rather simple structure with short loops connecting transmembrane helices II and III, and VI and VII (FIG. 12A). ECL2, which links helices IV and V, has a somewhat more extensive architecture that is unanticipated. In contrast to the buried, β-sheet structure of this loop in rhodopsin (FIG. 12B), ECL2 in β2AR is more exposed to the solvent and contains an extra helical segment. Additionally, there is an intra-loop disulfide bond between Cys184^(4.76) and Cys190^(5.29) that may help stabilize the more exposed ECL2. A second disulfide bond between Cys191^(5.30) and Cys106^(3.25) in helix III effectively ties ECL2 to the transmembrane core (Noda, et al., J Biol Chem 269, 6743 (1994)). The distal portion of ECL2 makes close contacts with ECL1 and contains a glycosylation site at Asn187^(5.26) (Mialet-Perez, et al., J Biol Chem 279, 38603 (2004)), which may serve to mask a grouping of aromatic residues on ECL1; in this construct, Asn1875.26 has been mutated to glutamate to aid in crystallization.

Electron density corresponding to the N-terminus was not apparent in the maps and, therefore, residues 1-28 are not included in the model. This disorder contrasts with rhodopsin, in which the N-terminus interacts extensively with the ECLs, forming a small four-strand β-sheet in conjunction with ECL2. This sheet structure forms a cap that effectively isolates the retinal binding site in a hydrophobic pocket (FIG. 13B). The lack of interactions between the N-terminus of β2AR and ECL2 further enables diffusible ligand access to the binding site. However a completely disordered N-terminus may be an artifact induced by the presence of the N-terminal Flag tag which carries an overall positive charge and may disrupt N-terminal interactions.

The short helical region on ECL2 adds a rigid structural element that, along with the two disulfide bonds, constrains the loop to a small range of conformations and helps stabilize the receptor by linking three transmembrane helices (FIG. 13A). This rigid conformation may help to stabilize the core of the receptor and lock ECL2 in a conformation that does not hinder access to the binding pocket.

Ligand Binding Site and Comparison to Rhodopsin

Carazolol is a partial inverse agonist that binds with picomolar affinity to β₂AR-T4L producing a reduction of the basal activity of the receptor. The crystal structure reveals extensive interactions between the receptor and carazolol that position the carbazole moiety adjacent to Phe289^(6.51), Phe290^(6.52), and Trp286^(6.48) (FIG. 13A, FIG. 7, and Table 5). In contrast, cis-retinal is a full inverse agonist covalently bound to rhodopsin, which suppresses all activity towards transducin (Palczewski, Annu Rev Biochem 75, 743 (2006)). Carazolol and retinal occupy similar spaces in their respective receptors, with significant overlap of the non-aromatic regions of carazolol. However, the β-ionone ring of retinal extends deep into the binding pocket of rhodopsin and contacts residues on helix V and VI, where it is sandwiched between Phe212^(5.47) and Tyr268^(6.51), and interacts with the highly conserved Trp265^(6.48) (FIG. 13B). It has been proposed that changes in the rotamer of Trp265^(6.48) occur upon activation of rhodopsin and related family members, and constitutes the “toggle switch” for receptor activation (Schwartz, et al., Annu Rev Pharmacol Toxicol 46, 481 (2006)). Accordingly, the interactions between c/s-retinal and Trp265^(6.48) are likely to contribute to the absence of basal activity in rhodopsin. Carazolol does not interact directly with the toggle switch on helix VI, however it lowers the basal activity of the receptor, and may do so by interacting with Phe289^(6.51) and Phe290^(6.52), which form an extended aromatic network surrounding the highly conserved Trp286^(6.48). As a result, Trp286^(6.48) adopts the rotamer associated with the inactive state. Thus, the steric constraints imposed by Phe290^(6.52) appear to structurally mimic the interaction of the β-ionone ring of retinal with the conserved Trp265^(6.48) and Phe212^(5.47) on rhodopsin (Shi et al., J Biol Chem 277, 40989 (2002)) (FIG. 13C).

TABLE 5 Direct contacts between β₂AR and carazolol. β₂AR atom Carazolol Distance (Å) Hydrogen Bond and Salt Bridge Contacts Asp113^(3.32) (OD2) N19 2.9 Asp113^(3.32) (OD1) O17 2.6 Ser203^(5.42) (OG) N7 3.2 Asn312^(7.39) (ND2) O17 2.9 Asn312^(7.39) (OD1) N19 2.9 Tyr316^(7.43) (OH) N19 3.4 Hydrophobic and Aromatic Interactions (closest distance for each residue, <4 Å) Trp109^(3.28) (CH2) C21 3.8 Val114^(3.33) (CG1) C11 3.9 Val117^(3.36) (CG1) C12 4.0 Thr118^(3.37) (OG1) C11 3.9 Phe193^(5.32) (CE2) C6 3.5 Tyr199^(5.38) (CE2) C2 3.9 Ser207^(5.46) (CB) C10 3.6 Trp286^(6.48) (CH2) O17 3.4 Phe289^(6.51) (CE2) O14 3.7 Phe290^(6.52) (CZ) C12 3.5 Asn293^(6.55) (ND2) C5 3.6 Tyr308^(7.35) (OH) C6 3.6

Structural Alignment and Helix Bundle Reorganization

It has long been thought that class A GPCRs share a similar architecture due to their predicted seven transmembrane helical bundles and sequence conservation within the membrane spanning regions (Lefkowitz, Nat Cell Biol 2, E133 (2000)). We aligned the structure of β2AR-T4L to highest resolution structure of rhodopsin (PDB ID Code 1U19) to evaluate the similarities and differences in ligand binding modes. We used difference distance matrices to select non-divergent areas between the two structures that align to reveal the differences in helix orientation between β2AR-T4L and rhodopsin. For the alignment, residues on β2AR were aligned to equivalent residues on Rhodopsin, respectively: 43-59 to 47-63; 67-95 to 71-99; 122-135 to 126-139; 285-296 to 264-275.

Relative to rhodopsin, the following helical shifts are seen in β2AR-T4L: the extracellular portions of helices I and III angle away from the center of the receptor, helix IV is translated away from the center of the receptor, helix V is translated closer to the center of the receptor and helix VI angles away from the receptor on the cytoplasmic end (FIG. 14). The largest difference is in helix I, which lacks a proline-induced kink found in rhodopsin and is comparatively straight. The angle between the rhodopsin and β2AR positions of helix I is approximately 18° with a shift of 7 Å at the apex on the extracellular face. This structural difference may arise from the need for an accessible binding site in β2AR, which is provided in part by a lack of interactions between the N-terminus and extracellular loop segments. In contrast the N-terminal region in rhodopsin occludes the retinal-binding site through extensive interactions with the extracellular loops (FIG. 12B). Helix V of β2AR is closer to the binding pocket by approximately 3.5 Å on average and its lumenal end is angled more towards helix VI. Helix IV of β2AR is further from the binding site, possibly to remove steric clashes resulting from the modified position of helix V (FIG. 14B, 14C). Helix III pivots further from the binding site about a fulcrum located close to the cytoplasmic end (FIG. 14C). The angle formed between rhodopsin helix III and the β2AR helix III is approximately 7°, yielding a 4 Å displacement out of the binding pocket at the cytoplasmic end of the helix. Helix VI is positioned further from the center of the receptor at the cytoplasmic end as compared to rhodopsin, which is caused by a slight difference in the angle about the proline-induced kink in the helix (FIG. 14C).

The ligand-binding pocket is formed by both structurally conserved and divergent helices as compared to rhodopsin (FIG. 14D). Helices III and V are two of the most conformationally shifted helices and contain the canonical catecholamine binding residues associated with activation of adrenergic family of receptors (Strader et al., J Biol Chem 263, 10267 (1988); Strader, et al., J Biol Chem 264, 13572 (1989); Liapakis et al., J Biol Chem 275, 37779 (2000)). The comparison with rhodopsin shows that the structurally conserved helices provide a common core present throughout the class A GPCRs, whereas the variable helices confer binding site plasticity with a resulting architecture capable of binding a large spectrum of ligands.

Comparison to Rhodopsin-Based GPCR Models

Since the determination of the inactive dark-state rhodopsin structure (Palczewski et al., Science 289, 739 (2000)), a number of homology models of other class A GPCRs have been reported (Bissantz, et al., Proteins 50, 5 (2003); Fano, et al., J Chem Inf Model 46, 1223 (2006); Hobrath, et al., J Med Chem 49, 4470 (2006); Nowak, et al., J Med Chem 49, 205 (2006); Zhang, et al., PLoS Comput Biol 2, e13 (2006)). Typically, homology models start by alignment of so-called fingerprint motifs that are common among the family. These fingerprint motifs are extrapolated to assign coordinates for the entire helical bundle. Loop regions are either ignored or modeled based on databases of loop conformations depending on the application (Bissantz, et. al, Proteins 50, 5 (2003)). A number of models exist for β2AR, some of which have been improved upon with supporting biochemical data (Bissantz, et. al, Proteins 50, 5 (2003); Zhang, et al., PLoS Comput Biol 2, e13 (2006); Freddolino et al., Proc Natl Acad Sci USA 101, 2736 (2004); Furse, et al., J Med Chem 46, 4450 (2003); Gouldson et al., Proteins 56, 67 (2004)). When compared to the β2AR structure reported here (according to the methods described above in this Example), however, all of these models were more similar to rhodopsin, as were models for other receptors (e.g. dopamine, muscarinic, and chemokine). This highlights a general shortcoming in homology models generated from a single structural template. The structural divergence between β2AR and rhodopsin would be quite difficult to predict accurately using only rhodopsin as a template.

Example 4 Structural Insights into β₂ Adrenergic Receptor Function

Methods

Molecular Biology for Generation of Mammalian and Sf9 Expression Constructs.

The insect cell expression plasmid that was used as a template for modification of the human β2AR gene has been described previously (Yao et al., Nat Chem Biol 2, 417 (2006)): the wild-type coding sequence of the human β2AR (starting at Gly2) was cloned into the pFastbacl Sf-9 expression vector (Invitrogen) with the HA signal sequence followed by the Flag epitope tag at the amino terminus and the third glycosylation site mutated as N187E. Using this template, a TAA stop codon was placed between Gly365 and Tyr366, terminating translation without the 48 C-terminal residues of the wild-type β2AR (“β2AR365”). A synthetic DNA cassette encoding the T4 Lysozyme (WT*-C54T, C97A) protein was made by overlapping extension PCR of 50-base oligonucleotides. This cassette was amplified and inserted into the β2AR365 construct between Ile233^(5.72) and Arg260^(6.22) (FIG. 21A), using the Quickchange Multi protocol (Stratagene). The corresponding mammalian cell expression plasmid was made by amplifying the entire fusion gene and cloning it into pcDNA3 (Invitrogen). Further deletions in the Sf9 and mammalian cell constructs were made using appropriate synthetic oligonucleotides in the Quickchange Multi protocol (Stratagene). All constructs were confirmed by sequencing.

HEK293 Cell Staining and Immunofluorescence Staining.

HEK293 cells were cultured on plastic dishes at 37° C. with 5% CO2 in Dulbecco's modified Eagle's medium (Cellgro) with 5% fetal bovine serum. For an individual expression experiment, cells at confluency were split, and approximately 100,000 cells were used to seed glass cover slips in the same medium. After 2 d, cells were transfected with the addition of 1 μg of a given pcDNA3-receptor plasmid and 3 μl of Fugene 6 reagent (Roche). 48 h after transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, blocked with PBS+2% goat serum, permeabilized with PBS+2% goat serum+0.5% Nonidet P-40 (Sigma), stained with Alexa488-conjugated M1 anti-FLAG antibody (for receptor) plus DAPI (nuclear) in blocking buffer, and washed with blocking buffer. Cover slips were mounted on microscope slides with Vectashield (Vector Labs) and dried overnight. Staining was visualized with an Axioplan 2 fluorescence imaging system, using a 63× objective and either green (Alexa488/FITC) or blue (DAPI/Hoechst) filter sets. A plasmid pcDNA3-β1AR, expressing an N-terminal FLAG-tagged β1 adrenergic receptor, was used as a positive control for cell-surface staining. Empty pcDNA3 was used as a negative control to assess background staining.

Expression and Purification of β₂AR-T4L from Baculovirus-Infected Sf9 Cells.

Recombinant baculovirus was made from pFastbac 1-β2AR-T4L using the Bac-to-Bac system (Invitrogen), as described previously (Yao et al., Nat Chem Biol 2, 417 (2006)). The β2AR-T4L protein was expressed in Sf9 insect cells infected with this baculovirus, and solubilized according to previously described methods (Kobilka, Anal Biochem 231, 269 (1995)). Dodecylmaltoside-solubilized receptor with the N-terminal FLAG epitope (DYKDDDA) (SEQ ID NO: 1) was purified by M1 antibody affinity chromatography (Sigma), treated with TCEP/iodoacetamide, and further purified by alprenolol-Sepharose chromatography (Kobilka, Anal Biochem 231, 269 (1995)) to isolate only functional GPCR. Eluted alprenolol-bound receptor was re-bound to M1 FLAG resin, and ligand exchange with 30 μM carazolol was performed on the column. β2AR-T4L was eluted from this final column with 0.2 mg/ml FLAG peptide in HLS buffer (0.1% dodecylmaltoside, 20 mM Hepes, 100 mM NaCl, pH 7.5) plus 30 μM carazolol and 5 mM EDTA. N-linked glycolsylations were removed by treatment with PNGaseF (NEB). Protein was concentrated from ˜5 mg/ml to 50 mg/ml with a 100 kDa molecular weight cut-off Vivaspin concentrator (Vivascience), and dialyzed against FILS buffer plus 10 μM carazolol.

Binding Measurements on Wild-Type β₂AR and β₂AR-T4L from Membranes.

Membrane preparation from baculovirus-infected Sf9 cells was performed as described previously (Swaminath, et al., Mol Pharmacol 61, 65 (2002)). For each binding reaction, membranes containing 0.7 μg total membrane protein were used. Saturation binding of [³H]-dihydroalprenolol (DHA) was measured by incubating membranes resuspended in 500 μl binding buffer (75 mM Tris, 12.5 mM MgCl₂, 1 mM EDTA, pH 7.4, supplemented with 0.4 mg/ml BSA) with 12 different concentrations of [³H]DHA (Perkin Elmer) between 20 pM and 10 nM. After 1 h incubation with shaking at 230 rpm, membranes were filtered from the binding reactions with a Brandel harvester, washed with binding buffer, and measured for bound [³H]DHA with a Beckman LS6000 scintillation counter. Non-specific binding was assessed by performing identical reactions in the presence of 1 μM alprenolol. For competition binding, membranes resuspended in 500 μl binding buffer were incubated with 0.5 nM [³H]DHA plus increasing concentrations of the competing ligand (all compounds were purchased from Sigma). For (−)-isoproterenol and (−)-epinephrine, concentrations were 100 pM-1 mM, each increasing by a factor of 10. For salbutamol, concentrations were 1 nM-10 mM. For ICI-118,551 and formoterol, concentrations were 1 pM-10 Non-specific binding was measured by using 1 μM unlabeled alprenolol as competing ligand. Each data point in the curves in FIGS. 2A and S1 represents the mean of three separate experiments, each done in triplicate. Binding data were analyzed by nonlinear regression analysis using Graphpad Prism. The values for K_(d) of [³H]DHA and K_(i) of other ligands are shown in Table 6.

TABLE 6 Saturation Binding [³H]DHA K_(d) ± SE (nM) Bmax (pmol/mg) β₂AR 0.161 ± 0.012 30.0 ± 0.5 β₂AR-T4L 0.180 ± 0.016 21.6 ± 0.5 Competition Binding K_(i) [S.E. interval] K_(i) [S.E. interval] Ligand for β₂AR (nM) for β₂AR-T4L (nM) (−)-isoproteronol  50.6 [48.9-52.3]  15.7 [15.2-16.2] (−)-epinephrine   175 [163-188]  56.0 [52.8-59.4] salbutamol   728 [708-750]   307 [291-323] ICI-118,551 0.617 [0.570-0.668] 0.626 [0.591-0.662] formoterol  3.60 [3.39-3.83]  1.68 [1.55-1.81] Binding affinities of different ligands for the wild-type β₂AR and the fusion protein β₂AR-T4L. The saturation and competition binding curves shown in FIG. 22 were fit to theoretical saturation and one-site competition binding models, using the program Graphpad Prism. K_(i) values were calculated using the Cheng-Prusoff equation: K_(i) = IC₅₀/(1 + [ligand]/K_(d)).

Bimane Fluorescence Experiments on Purified, Detergent-Solubilized Receptors

β₂AR-T4L and β₂AR365 were purified as described above, with two important differences. First, prior to iodoacetamide treatment, FLAG-pure receptor at 2.5 μM (measured by soluble [³H]DHA binding) was incubated with 5 μM monobromobimane for 1 h at 4° C. Second, after binding the bimane-labeled alprenolol-Sepharose-purified receptor to M1 antibody resin, the column was washed extensively with ligand-free buffer before elution. Based on previous precedent (Ghanouni, et al., Proc Natl Acad Sci USA 98, 5997 (2001)), this protocol is expected to target primarily Cys265^(6.27) for fluorophore derivitization. Fluorescence spectroscopy was performed on a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) with photon-counting mode, using an excitation and emission bandpass of 5 nm. All experiments were done at 25° C. For emission scans, we set excitation at 350 nm and measured emission from 417 to 530 nm with an integration time of 1.0 s nm⁻¹. To determine the effect of ligands, spectra were measured after 15 min incubation with different compounds (at saturating concentrations: [(−)-isoproterenol]=100 μM; [ICI-118,551]=10 μM; [salbutamol]=500 μM). Fluorescence intensity was corrected for background fluorescence from buffer and ligands in all experiments. The curves shown in FIG. 22B are each the average of triplicate experiments performed in parallel. λ_(max) values and intensity changes for β₂AR-T4L and β₂AR365, each incubated with different ligands, are tabulated in Table 7.

TABLE 7 λmax ± SD for λmax ± SD for Ligand (β₂AR365 (nm) β₂AR-T4L (nm) none 448 ± 2 447 ± 2 (−)-isoproteronol 453 ± 2 455 ± 2 ICI-118,551 447 ± 1 446 ± 1 salbutamol 449 ± 1 449 ± 1 Intensity λmax_(Ligand) /Intensity λmax_(none) Ligand β₂AR365 β₂AR-T4L (−)-isoproteronol 0.758 ± 0.007 0.824 ± 0.006 ICI-118,551 1.013 ± 0.008 1.028 ± 0.008 salbutamol 0.950 ± 0.013 0.928 ± 0.009 Bimane fluorescence responses for unliganded β₂AR365 and β₂AR-T4L, incubated for 15 min with different ligands. Top panel shows the λ_(max) for fluorescence emission spectra (excitation at 350 nm and emission from 417 to 530 nm) collected after 15 min incubation with ligand. Each value is mean ± standard deviation for triplicate experiments performed in parallel. Bottom panel shows the change in fluorescence intensity after incubation with ligand, represented as the ratio of Intensity λmax of the ligand to Intensity at λ_(max) of the control no ligand (“none”) response.

Comparing the Proteolytic Stability of Unliganded β₂AR and β₂AR-T4L.

The limited trypsin proteolysis protocol was adapted from Jiang et al., Biochemistry 44, 1163 (2005). Carazolol-bound β₂AR-T4L or wild-type β₂AR (each at 30 mg/ml) were diluted 10-fold into HLS buffer (see above) and TPCK-trypsin was added at a 1:1000 ratio (wt:wt). The digests were incubated at room temperature. At various time points, aliquots were removed and flash frozen on dry ice/ethanol. After the last aliquot was removed, all samples were thawed, and an equal volume of 10% SDS/PAGE loading buffer was added to each. Samples were then analyzed by electrophoresis on 12% polyacrylamide gels, followed by staining with Coomassie blue (FIG. 16).

Comparing the Stability of Unliganded β₂AR and β₂AR-T4L

Unliganded β2AR365 and β2AR-T4L were each purified as described above for the bimane experiments. 200 μl 0.02 mg/ml receptor in HLS buffer was incubated at 37° C. on a heating block. At the time points indicated in FIG. 17, samples were briefly spun and gently vortexed and 16.5 μl was removed and diluted 18.2-fold in HLS (300 μl total). Then 4×5 μl was removed for determination of total binding and 2×5 μl was removed for nonspecific binding. To measure soluble binding, 5 μl diluted receptor was added to 105 μl HLS (400-fold final dilution of receptor) containing 10 nM [3H]DHA±10 μq cold alprenolol. Reactions were incubated 30 min at RT, then on ice until processing. 100 μl of each reaction was applied to a 1 ml G50 column to separate protein from residual unbound [3H]DHA, and receptor was eluted using 1.1 ml ice-cold HLS. Bound [3H]DHA was quantified on a Beckman LS6000 scintillation counter.

Carazolol Dissociation from the “Wild-Type” Receptor β₂AR365

β2AR365 was purified with carazolol bound, according to the protocol described above for β2ART4L. Carazolol-bound receptor (at approximately 50 μM concentration) was dialyzed in the dark against 1 L dialysis buffer (20 mM HEPES pH7.5, 100 mM NaCl, 0.1% dodecylmaltoside, 300 micromolar alprenolol) at room temperature with stirring. At indicated time points, two samples were removed from the parafilm-sealed open-ended dialysis chamber, diluted into fresh dialysis buffer, and carazolol emission spectra were obtained on a Spex FluoroMax spectrofluorometer (using excitation at 330 nm and emission from 335 to 400 nm). As internal standards for every time point, samples were removed for determination of protein concentration using the Bio-Rad Protein DC kit (FIG. 19).

CAM and UCM Mutants

The CAMs (constitutively active mutants) described in the literature that are the basis for FIG. 26A and the associated discussion are: L124A (Tao, et al., Mol Endocrinol 14, 1272 (2000)), C116F (Zuscik, et al., J Biol Chem 273, 3401 (1998)), D130A (Rasmussen et al., Mol Pharmacol 56, 175 (1999)), L272C (Jensen et al., J Biol Chem 276, 9279 (2001)), and C285T (Shi et al., J Biol Chem 277, 40989 (2002)). The UCMs (uncoupling mutations) from the literature that were used for FIG. 26C are: D79N (Chung, et al., J Biol Chem 263, 4052 (1988)), F139A (Moro, et al., J Biol Chem 269, 6651 (1994)), T1641 (Green, et al., J Biol Chem 268, 23116 (1993)), N318K (Strader et al., Proc Natl Acad Sci USA 84, 4384 (1987)), N322A (Barak, et al., Biochemistry 34, 15407 (1995)), P323A (Barak, et al., Biochemistry 34, 15407 (1995)), Y326A (Barak, et al., Biochemistry 34, 15407 (1995)), L339A (Gabilondo et al., Proc Natl Acad Sci USA 94, 12285 (1997)), and L340A (Gabilondo et al., Proc Natl Acad Sci USA 94, 12285 (1997)).

Biochemical and Structural Analysis of β₂AR-T4L

The β₂AR fusion protein in which T4 Lysozyme replaces most of the third intracellular loop of the GPCR (“β₂AR-T4L”) retains near-native pharmacologic properties. The β₂AR-T4L protein was crystallized in lipidic cubic phase, as described in the Examples above, and the resulting 2.4 Å resolution crystal structure reveals the interface between the receptor and the ligand carazolol, a partial inverse agonist. The efficacy of a ligand describes the effect of the ligand on the functional properties of a GPCR. For purposes of the Examples only, agonists are defined as ligands that fully activate the receptor; partial agonists induce submaximal activation even at saturating concentrations; inverse agonists inhibit basal receptor activity, and antagonists have no effect on basal activity, but competitively block access of other ligands. Carazolol, is defined as a partial inverse agonist because it suppresses only 50% of the basal activity of the β₂AR. Analysis of mutagenesis data in light of the structure clarifies the roles of different amino acids in inverse agonist binding, and implies that rearrangement of the binding pocket accompanies agonist binding. In addition, the structure reveals how mutations known to cause constitutive activity or uncoupling of agonist binding and G-protein activation are distributed between the ligand-binding pocket and the cytoplasmic surface of the protein, such that changes in side chains due to interaction with the ligand can be transmitted through the structure to the site of G protein interaction.

Cloning of β₂AR-T4L

DNA encoding the T4L protein (C54T, C97A) (Matsumura, et al., Proc Natl Acad Sci USA 86, 6562 (1989)) was initially cloned into the human β2AR gene, guided by comparison of ICL3 length and sequence among class A GPCRs (Horn et al., Nucleic Acids Res 31, 294 (2003)): residues 234^(5.73)-259^(6.21) of the β2AR were replaced by residues 2-164 of T4L (construct “E3” in FIG. 21A). In addition, the receptor was truncated at position 365, which aligns approximately with the position of the rhodopsin carboxyl terminus. Although these modifications resulted in a receptor that was expressed efficiently in Sf9 cells, further optimization was carried out to reduce the length of the junction between the receptor and the T4L termini, as described in the methods above. Several candidate constructs are illustrated in FIG. 21A, and selected immunofluorescence images of transfected, permeabilized HEK293 cells are shown in FIG. 21B. Relative to the initial construct, we could remove three residues from the cytoplasmic end of helix V, three residues from the C-terminal end of T4L, and three residues from the N terminus of helix VI, all without losing significant cell-surface expression. The final construct used for crystallization trials (“β2AR-T4L”) has residues 231^(5.70)-262^(6.24) of the β2AR replaced by amino acids 2-161 of T4L (“1D” in FIG. 21A). Similar reduction of flexibility through minimization of linker length has been important in previous crystallization studies on soluble fusion proteins (Smyth, et al., Protein Sci 12, 1313 (2003)).

Functional Properties of β₂AR-T4L

We measured saturation binding of [3H]DHA to the β2AR-T4L, as well as competition binding of the inverse agonist ICI-118,551 and several agonists (FIG. 22A, FIG. 15 and Table 6). The results show that β2AR-T4L has wild-type affinity for the antagonist [3H]DHA and the inverse agonist ICI-118,551, whereas the affinity for both agonists (isoproterenol, epinephrine, formoterol) and a partial agonist (salbutamol) is two to three-fold higher relative to wild-type β2AR. Higher agonist binding affinity is a property associated with constitutively active mutants (CAMs) of GPCRs. CAMs of the β2AR also exhibit elevated basal, agonist-independent activation of Gs, and typically have lower expression levels and reduced stability (Gether et al., J Biol Chem 272, 2587 (1997); Rasmussen et al., Mol Pharmacol 56, 175 (1999)). β2ART4L exhibits binding properties of a CAM, but it expresses at levels exceeding 1 mg per liter of Sf9 cell culture, is more resistant to trypsin proteolysis than the wild-type β2AR (FIG. 16), and retains binding activity in detergent at 37° C. as well as the wild-type receptor (FIG. 17).

β2AR-T4L did not couple to Gs, as expected due to the replacement of ICL3 by T4L. To assess whether the fused protein alters receptor function at the level of its ability to undergo conformational changes, we used a covalently attached fluorescent probe as a reporter for ligand-induced structural changes. Fluorophores attached at Cys265^(6.27), at the cytoplasmic end of helix VI, detect agonist-induced conformational changes that correlate with the efficacy of the agonist towards G protein activation (Ghanouni et al., J Biol Chem 276, 24433 (2001); Ghanouni, et al., Proc Natl Acad Sci USA 98, 5997 (2001); Swaminath et al., J Biol Chem 279, 686 (2004); Swaminath et al., J Biol Chem 280, 22165 (2005)). Detergent-solubilized β2AR365 (wild-type receptor truncated at 365) and β2AR-T4L were each labeled with monobromobimane. Addition of the agonist isoproterenol to purified β2AR365 induces a decrease in fluorescence intensity and a shift in λmax for the attached bimane probe (FIG. 22B and Table 7). These changes in intensity and λmax are consistent with an agonist-induced increase in polarity around bimane. A smaller change is observed with the partial agonist salbutamol, while the inverse agonist ICI-118,551 had little effect. For the β2AR-T4L, there are subtle differences in the baseline spectrum of the bimane-labeled fusion protein, as might be expected if the environment around Cys265^(6.27) is altered by T4L. However, the full agonist isoproterenol induces a qualitatively similar decrease in intensity and rightward shift in Amax. Thus the presence of the fused T4L does not prevent agonist-induced conformational changes. The partial agonist salbutamol induced larger responses in β2AR-T4L than were observed in wild-type β2AR, and there was a small increase in fluorescence in response to the inverse agonist ICI-118,551. These are properties observed in CAMs (Gether et al., J Biol Chem 272, 2587 (1997); Samama, et al., J Biol Chem 268, 4625 (1993)) and are consistent with the higher affinities for agonists and partial agonists exhibited by β2AR-T4L. Therefore, we conclude that the T4L fusion induces a partial constitutively active phenotype in the β2AR, likely caused by changes at the cytoplasmic ends of helices V and VI.

Comparison Between β₂AR-T4L and β₂AR-Fab Structures

The β₂AR-T4L fusion strategy is validated by comparison of its structure to the structure of wild-type β₂AR complexed with a Fab that recognizes a three dimensional epitope consisting of the amino and carboxyl-terminal ends of ICL3, determined at an anisotropic resolution of 3.4 Å/3.7 Å (Rasmussen et al., Nature, 7168:355-6 (2007)). FIG. 23A illustrates the similarity between the fusion and antibody complex approaches to β₂AR crystallization, in that both strategies rely on attachment (covalent or non-covalent, respectively) of a soluble protein partner between helices V and VI. A major difference between the two structures is that the extracellular loops and the carazolol ligand could not be modeled in the β₂AR-Fab complex, whereas these regions are resolved in the structure of β₂AR-T4L. Nonetheless, it is clear that the T4L insertion does not significantly alter the receptor. Superposition of the two structures (FIG. 18) illustrates that the transmembrane helices of the receptor components are very similar (RMSD=0.8 Å for 154 common modeled transmembrane Ca positions, versus 2.3 Å between β₂AR-T4L and the 154 equivalent residues in rhodopsin), especially when the modest resolution of the Fab complex is taken into account.

There is one significant difference between the Fab-complex and chimeric receptor structures that can be attributed to the presence of T4L. The cytoplasmic end of helix VI is pulled outward as a result of the fusion to the carboxyl terminus of T4L, which alters the packing of Phe264^(6.26) at the end of helix VI (FIG. 23B). In the Fab-complex β₂AR, interactions between Phe264^(6.26) and residues in helix V, helix VI, and ICL2 may be important in maintaining the β₂AR in the basal state. The loss of these packing interactions in β₂AR-T4L could contribute to the higher agonist binding affinity characteristic of a CAM.

An unexpected difference between the structure of rhodopsin and the β₂AR-T4L involves the sequence E/DRY found at the cytoplasmic end of helix III in 71% of class A GPCRs. In rhodopsin, Glu134^(1.49) and Arg135^(3.50) form a network of hydrogen bond and ionic interactions with Glu247^(63.0) at the cytoplasmic end of helix VI. These interactions have been referred to as an “ionic lock” that stabilizes the inactive state of rhodopsin and other class A members (Ballesteros et al., J Biol Chem 276, 29171 (2001)). However, the arrangement of the homologous residues is significantly different in β₂AR-T4L: Arg131^(3.50) interacts primarily with Asp130^(3.49) and a sulfate ion rather than with Glu268^(6.30), and the distance between helix III and helix VI is greater than in rhodopsin (FIG. 23C). The fact that similar ionic lock structures were obtained using two different approaches suggests that a broken ionic lock is a genuine feature of the carazolol-bound state of the receptor.

Ligand Binding to the β₂AR

The β₂AR-T4L fusion protein was purified and crystallized in complex with the inverse agonist carazolol. Carazolol stabilizes the β₂AR against extremes of pH and temperature, perhaps related to its unusually high binding affinity (K_(d)<0.1 nM) and slow dissociation kinetics (t_(1/2)˜30 h) (FIG. 19). The interactions between carazolol and β₂AR-T4L are depicted schematically in FIG. 24. The carbazole ring system is oriented roughly perpendicular to the plane of the membrane, and the alkylamine chain (atoms 15-22 in the model) is nearly parallel to the heterocycle (FIG. 25A-B). As described in Example 3, above, carazolol was modeled into the electron density as the (S)-(−) isomer due to the higher affinity of this enantiomer, despite the fact that a racemic mixture of the ligand was used in crystallization. Asp113^(3.32), Tyr316^(7.43), and Asn312^(7.39) present a constellation of polar functional groups to the alkylamine and alcohol moieties of the ligand, with Asp113^(3.32) and Asn312^(7.39) sidechains forming close contacts (<3 Å) with O₁₇ and N₁₉ atoms of carazolol (FIGS. 24 and 25A-B). Asp113^(3.32) was one of the first β₂AR residues shown to be important for ligand binding; notably the D113N mutation causes complete loss of detectable affinity for antagonists (Strader et al., Proc Natl Acad Sci USA 84, 4384 (1987)) and a decrease in the potency of agonists towards cell-based G protein activation by over 4 orders of magnitude (Strader et al., J Biol Chem 263, 10267 (1988)). Likewise, mutations of Asn312^(7.39) perturb β₂AR binding to agonists and antagonists: changes to nonpolar amino acids (Ala or Phe) reduce affinities to undetectable levels, while retention of a polar functionality (Thr or Gln) gives partial affinity (Suryanarayana, et al., Mol Pharmacol 44, 111 (1993)). On the opposite end of the ligand near helix V, N₇ of the carbazole heterocycle forms a hydrogen bond with the side chain hydroxyl of Ser203^(5.42). Interestingly, mutations of Ser203^(5.42) specifically decrease β₂AR affinity towards catecholamine agonists and aryloxyalkylamine ligands with nitrogen-containing heterocycles such as pindolol (Liapakis et al., J Biol Chem 275, 37779 (2000)), and by implication carazolol. Thus, the polar interactions between carazolol and the receptor observed in the crystal structure agree with the known biochemical data. The contribution of Tyr316^(7.43) to antagonist and agonist affinity remains to be tested; this residue is conserved as tyrosine in all sequenced adrenergic receptor genes (Horn et al., Nucleic Acids Res 31, 294 (2003)).

FIG. 25C shows the tight packing between carazolol and surrounding amino acids that buries 790 Å² of surface area from solvent; specific contacts are depicted schematically in FIG. 24. Notable among the hydrophobic residues contacting carazolol are Val114^(3.33), Phe290^(6.52), and Phe193^(5.32). The side chain of Val114^(3.33) from helix III makes multiple contacts with the C₈-C₁₃ ring of the carbazole heterocycle, and Phe290^(6.52) from helix VI forms an edge-to-face aromatic interaction with the same ring. As a result, these two amino acids form a hydrophobic “sandwich” with the portion of the aryl moiety that is common to many adrenergic antagonists. Mutation of Val114^(3.33) to alanine was shown to decrease β₂AR affinity towards the antagonist alprenolol by an order of magnitude, as well as lowering affinity for the agonist epinephrine 300-fold (P. Chelikani et al., Proc Natl Acad Sci USA 104, 7027 (2007)). Phe193^(5.32) is different from other carazolol contact residues in that it is located on the ECL2, in the path of hormone accessibility to the binding pocket. This amino acid contributes more buried surface area than any other residue to the interface between β₂AR-T4L and carazolol (see Table 8). Therefore, Phe193^(5.32) is likely to contribute significantly to the energy of β₂AR-carazolol complex formation, and the position of this residue on the extracellular side of the binding site may allow it to act as a gate that contributes to the unusually slow dissociation of the ligand (FIG. 19).

TABLE 8 β₂AR residue Surface area buried (Å²) Trp109^(3.28) 21.4 Thr110^(3.29) 5.7 Asp113^(3.32) 19.3 Val114^(3.33) 25.5 Val117^(3.36) 8.5 Thr118^(3.37) 1.9 Phe193^(5.32) 51.2 Thr195^(5.34) 7.4 Tyr199^(5.38) 7.6 Ala200^(5.39) 10.0 Ser203^(5.42) 9.0 Ser204^(5.43) 4.6 Ser207^(5.46) 6.3 Trp286^(6.48) 3.1 Phe289^(6.51) 20.0 Phe290^(6.52) 19.0 Phe293^(6.55) 18.7 Tyr308^(7.35) 14.4 Asn312^(7.39) 22.5 Tyr316^(7.43) 6.5 Buried surface area contributions at the β₂AR-T4L/carazolol interface. Solvent accessible surface area calculations were done with the CNS software package (Brunger et al., Acta Crystallogr D Biol Crystallogr 54, 905 (1998)), using a probe radius of 1.4 Å. Buried surface area contributions of individual residues were determined by calculating solvent-accessible surface area per residue for the full β₂ART4L/carazolol model, and subtracting these numbers from the calculated values for the receptor model without carazolol.

Analysis of the binding pocket provides insights into the structural basis for pharmacologic selectivity between the β₂AR and closely related adrenergic receptors such as the β₁AR. The affinities of these two receptors for certain ligands, such as ICI-118,551, betaxolol and RO363 (Sugimoto et al., J Pharmacol Exp Ther 301, 51 (2002)), differ by up to 100-fold. Curiously, all of the amino acids in the carazolol binding pocket are conserved between the β₁AR and β₂AR (see FIG. 20). The majority of the 94 amino acid differences between the β₁AR and β₂AR are found in the cytoplasmic and extracellular loops. While residues that differ in the transmembrane segments generally face the lipid bilayer, eight residues lie at the interface between helices and may influence helix packing. The structural basis for pharmacologic differences between β₁AR and β₂AR must, therefore, arise from amino acid differences in the entrance to the binding pocket or subtle differences in the packing of helices. Evidence for the latter comes from chimeric receptor studies (Frielle, et al., Proc Natl Acad Sci USA 85, 9494 (1988)) in which successive exchange of helices between PAR and β₂ARs led to a gradual change in affinity for the β₂AR selective ICI-118,551 and the β₁AR selective betaxolol.

As discussed above, β₂AR-T4L shows CAM-like properties with respect to agonist binding affinities, suggesting that the unliganded β₂AR-T4L may exist in a more active conformation than the wild type-β₂AR. Nevertheless, as shown in FIG. 22B, β₂AR-T4L can be stabilized in an inactive conformation by an inverse agonist. Since β₂AR-T4L was crystallized with bound carazolol, a partial inverse agonist, the structure most likely represents an inactive state. This is consistent with the similarity of the β₂AR-T4L and β₂AR-Fab5 carazolol-bound structures. To assess whether conformational changes are required to accommodate catecholamines, a model of isoproterenol was placed in the binding site such that common atoms (16-22 in FIG. 24) were superimposed onto the analogous carazolol coordinates in the crystal structure (FIG. 25D). Residues Ser204^(5.43) and Ser207^(5.46) are critical for catecholamine binding and activation of the β₂AR, with Ser204^(5.43) hydrogen bonding to the meta-hydroxyl and Ser207^(5.46) to the para-hydroxyl of the catechol ring, respectively (Strader, et al., J Biol Chem 264, 13572 (1989)). In our model, the catechol hydroxyls of isoproterenol face the appropriate serines on helix V, but the distances are too long for hydrogen bonding (6.8 Å from meta-hydroxyloxygen to the sidechain oxygen of Ser204^(5.43), 4.8 Å from the para-hydroxyloxygen to the sidechain oxygen of Ser207^(5.46)). In addition, Asn293^(6.55) and Tyr308^(7.35), two residues expected to form selective interactions with agonists based on the literature (Wieland, et al., Proc Natl Acad Sci USA 93, 9276 (1996); Kikkawa, et al., Mol Pharmacol 53, 128 (1998)), are too distant to form productive polar or hydrophobic contacts with the modeled isoproteronol molecule. These observations suggest that agonist binding requires changes in the binding site relative to the carazolol-bound structure, unless common structural components of agonists and inverse agonists bind in a significantly different manner.

Structural Insights into β₂AR Activation

Analysis of mutations that affect β₂AR function provides insights into structural rearrangements that are likely to occur during receptor activation. FIG. 26A illustrates the location of amino acids for which mutations lead to elevated basal, agonist-independent activity (constitutively active mutations, CAMs), as well as amino acids for which mutations impair agonist activation (uncoupling mutations, UCMs). Residues for which CAMs have been described are likely to be involved in interactions that maintain the receptor in the inactive conformation. These amino acids are centrally located on helices III and VI. In contrast, positions in which UCMs have been observed are likely to form intramolecular interactions that stabilize the active state. A cluster of UCMs are found at the cytoplasmic end of helix VII. Neither CAMs nor UCMs are directly involved in agonist binding. Although the CAMs and UCMs are not directly connected in sequence, it is evident from the structure that they are linked through packing interactions, such that movements in one will likely affect the packing of others. For example, FIG. 26A (right panel) shows all amino acids with atoms within 4 Å of the two centrally located CAMs, Leu124^(3.43) (Tao, et al., Mol Endocrinol 14, 1272 (2000)) and Leu272^(6.34) (Jensen et al., J Biol Chem 276, 9279 (2001)). Several amino acids that pack against these CAMs also interact with one or more UCMs. Trp286^(6.48) lies at the base of the binding pocket. It has been proposed that agonist binding leads to a change in the rotameric state of Trp286^(6.48) with subsequent changes in the angle of the helical kink formed by Pro288^(6.50) (Shi et al., J Biol Chem 277, 40989 (2002)). It is likely that an agonist-induced change in the rotameric state of Trp286^(6.48) will be linked to changes in sidechains of CAMs and UCMs through packing interactions and propagated to the cytoplasmic ends of the helices and the associated intracellular loops that interact with G proteins and other signaling molecules.

In the structures of both rhodopsin and the β₂AR, a cluster of water molecules lies near the most highly conserved class A GPCR residues (FIG. 26B). It has been proposed that these water molecules may play a role in the structural changes involved in receptor activation (Pardo, et al., Chembiochem 8, 19 (2007)). FIG. 26C shows the network of potential hydrogen bonding interactions that link Trp286^(6.48) with conserved amino acids extending to the cytoplasmic ends of helices. UCMs have been identified for three amino acids linked by this network—N322^(7.49), P323^(7.50), and Y326^(7.33) (Barak et al., Biochemistry 34, 15407 (1995)). This relatively loose-packed, water filled region is likely to be important in allowing conformational transitions, as there will be fewer steric restraints to sidechain repacking.

All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

APPENDIX 1 HEADER  MEMBRANE PROTEIN/HYDROLASE    05-OCT-07  2RH1 TITLE  HIGH RESOLUTION CRYSTAL STRUCTURE OF HUMAN B2-ADRENERGIC G TITLE 2 PROTEIN-COUPLED RECEPTOR COMPND  MOL_ID: 1; COMPND  2 MOLECULE: BETA-2-ADRENERGIC RECEPTOR/T4-LYSOZYME CHIMERA; COMPND  3 CHAIN: A; COMPND  4 SYNONYM: BETA-2 ADRENERGIC RECEPTOR, BETA-2 ADRENOCEPTOR, COMPND  5 BETA-2 ADRENORECEPTOR/LYSIS PROTEIN, MURAMIDASE, COMPND  6 ENDOLYSIN; COMPND  7 ENGINEERED: YES; COMPND  8 MUTATION: YES SOURCE   MOL_ID: 1; SOURCE  2 ORGANISM_SCIENTIFIC: HOMO SAPIENS, ENTEROBACTERIA PHAGE T4; SOURCE  3 ORGANISM_COMMON: HUMAN,; SOURCE  4 STRAIN:,; SOURCE  5 GENE: ADRB2, ADRB2R, B2AR/E; SOURCE  6 EXPRESSION_SYSTEM: SPODOPTERA FRUGIPERDA; SOURCE  7 EXPRESSION_SYSTEM_COMMON: FALL ARMYWORM; SOURCE  8 EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID; SOURCE  9 EXPRESSION_SYSTEM_PLASMID: PFASTBAC1; SOURCE 10 OTHER_DETAILS: THE CONSTRUCT HAS BEEN OBTAINED BY SOURCE 11 OVERLAPPING EXTENSION PCR KEYWDS   GPCR, 7TM, ADRENERGIC, FUSION, LIPIDIC CUBIC PHASE, LIPIDIC, KEYWDS  2 MESOPHASE, CHOLESTEROL, MEMBRANE PROTEIN, MEMBRANE PROTEIN KEYWDS  3 / HYDROLASE COMPLEX EXPDTA  X-RAY DIFFRACTION AUTHOR  V. CHEREZOV, D. M. ROSENBAUM, M. A. HANSON, S. G. F. RASMUSSEN, AUTHOR  2 F. S. THIAN, T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS, B. K. KOBILKA, AUTHOR  3 R. C. STEVENS REVDAT 5  07-OCT-08 2RH1 1 REMARK REVDAT 4  11-DEC-07 2RH1 1 REMARK REVDAT 3  04-DEC-07 2RH1 1 JRNL REVDAT 2  06-NOV-07 2RH1 1 JRNL  HELIX SHEET REVDAT 1  30-OCT-07 2RH1 0 JRNL AUTH  V. CHEREZOV, D. M. ROSENBAUM, M. A. HANSON, S. G. RASMUSSEN, JRNL AUTH 2 F. S. THIAN, T. S. KOBILKA, H. J. CHOI, P. KUHN, W. I. WEIS, JRNL AUTH 3 B. K. KOBILKA, R. C. STEVENS JRNL TITL  HIGH-RESOLUTION CRYSTAL STRUCTURE OF AN ENGINEERED JRNL TITL 2 HUMAN BETA2-ADRENERGIC G PROTEIN-COUPLED RECEPTOR. JRNL REF  SCIENCE      V. 318 1258 2007 JRNL REFN  ASTM SCIEAS US ISSN 0036-8075 REMARK 1 REMARK 1 REFERENCE 1 REMARK 1  AUTH  D. M. ROSENBAUM, V. CHEREZOV, M. A. HANSON, REMARK 1  AUTH 2 S. G. F. RASMUSSEN, F. S. THIAN, T. S. KOBILKA, H. J. CHOI, REMARK 1  AUTH 3 X. J. YAO, W. I. WEIS, R. C. STEVENS, B. K. KOBILKA REMARK 1  TITL  GPCR ENGINEERING YIELDS HIGH-RESOLUTION STRUCTURAL REMARK 1  TITL 2 INSIGHTS INTO BETA2 ADRENERGIC RECEPTOR FUNCTION. REMARK 1  REF  TO BE PUBLISHED REMARK 1  REFN REMARK 2 REMARK 2 RESOLUTION. 2.40 ANGSTROMS. REMARK 3 REMARK 3 REFINEMENT. REMARK 3  PROGRAM  : REFMAC 5.2.0019 REMARK 3  AUTHORS  : MURSHUDOV, VAGIN, DODSON REMARK 3 REMARK 3  REFINEMENT TARGET: MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3  RESOLUTION RANGE HIGH (ANGSTROMS): 2.40 REMARK 3  RESOLUTION RANGE LOW (ANGSTROMS): 19.95 REMARK 3  DATA CUTOFF    (SIGMA(F)): 0.000 REMARK 3  COMPLETENESS FOR RANGE   (%): 99.8 REMARK 3  NUMBER OF REFLECTIONS    : 26506 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3  CROSS-VALIDATION METHOD   : THROUGHOUT REMARK 3  FREE R VALUE TEST SET SELECTION  : RANDOM REMARK 3  R VALUE  (WORKING + TEST SET): 0.198 REMARK 3  R VALUE    (WORKING SET): 0.196 REMARK 3  FREE R VALUE      : 0.232 REMARK 3  FREE R VALUE TEST SET SIZE  (%): 4.900 REMARK 3  FREE R VALUE TEST SET COUNT  : 1310 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3  TOTAL NUMBER OF BINS USED   : 20 REMARK 3  BIN RESOLUTION RANGE HIGH   : 2.40 REMARK 3  BIN RESOLUTION RANGE LOW    : 2.46 REMARK 3  REFLECTION IN BIN (WORKING SET): 1829 REMARK 3  BIN COMPLETENESS (WORKING + TEST) (%): 98.76 REMARK 3  BIN R VALUE   (WORKING SET): 0.2700 REMARK 3  BIN FREE R VALUE SET COUNT   : 78 REMARK 3  BIN FREE R VALUE     : 0.3010 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3  ALL ATOMS    : 3804 REMARK 3 REMARK 3 B VALUES. REMARK 3  FROM WILSON PLOT   (A**2): 63.91 REMARK 3  MEAN B VALUE  (OVERALL, A**2): 63.91 REMARK 3  OVERALL ANISOTROPIC B VALUE. REMARK 3  B11 (A**2): 0.43000 REMARK 3  B22 (A**2): −3.46000 REMARK 3  B33 (A**2): 4.25000 REMARK 3  B12 (A**2): 0.00000 REMARK 3  B13 (A**2): 2.26000 REMARK 3  B23 (A**2): 0.00000 REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3  ESU BASED ON R VALUE       (A): 0.295 REMARK 3  ESU BASED ON FREE R VALUE      (A): 0.220 REMARK 3  ESU BASED ON MAXIMUM LIKELIHOOD     (A): 0.203 REMARK 3  ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 18.501 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3  CORRELATION COEFFICIENT FO-FC  : 0.963 REMARK 3  CORRELATION COEFFICIENT FO-FC FREE: 0.944 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES   COUNT  RMS  WEIGHT REMARK 3  BOND LENGTHS REFINED ATOMS   (A): 3843; 0.013; 0.022 REMARK 3  BOND LENGTHS OTHERS    (A): 2622; 0.000; 0.020 REMARK 3  BOND ANGLES REFINED ATOMS  (DEGREES): 5219; 1.500; 2.000 REMARK 3  BOND ANGLES OTHERS   (DEGREES): 6377; 4.099; 3.002 REMARK 3  TORSION ANGLES, PERIOD 1  (DEGREES):  441; 3.876; 5.000 REMARK 3  TORSION ANGLES, PERIOD 2  (DEGREES):  154; 31.604; 23.182 REMARK 3  TORSION ANGLES, PERIOD 3  (DEGREES):  627; 11.383; 15.000 REMARK 3  TORSION ANGLES, PERIOD 4  (DEGREES):   22; 12.164; 15.000 REMARK 3  CHIRAL-CENTER RESTRAINTS   (A**3):  610; 0.060; 0.200 REMARK 3  GENERAL PLANES REFINED ATOMS  (A): 4008; 0.001; 0.020 REMARK 3  GENERAL PLANES OTHERS   (A):  804; 0.001; 0.020 REMARK 3  NON-BONDED CONTACTS REFINED ATOMS (A):  926; 0.187; 0.300 REMARK 3  NON-BONDED CONTACTS OTHERS   (A):  2443; 0.194; 0.300 REMARK 3  NON-BONDED TORSION REFINED ATOMS (A):  1935; 0.180; 0.500 REMARK 3  NON-BONDED TORSION OTHERS   (A):  1580; 0.107; 0.500 REMARK 3  H-BOND (X . . . Y) REFINED ATOMS  (A):  194; 0.164; 0.500 REMARK 3  H-BOND (X . . . Y) OTHERS   (A): NULL; NULL; NULL REMARK 3  POTENTIAL METAL-ION REFINED ATOMS  (A): NULL; NULL; NULL REMARK 3  POTENTIAL METAL-ION OTHERS   (A): NULL; NULL; NULL REMARK 3  SYMMETRY VDW REFINED ATOMS   (A):  7; 0.155; 0.300 REMARK 3  SYMMETRY VDW OTHERS    (A):  29; 0.140; 0.300 REMARK 3  SYMMETRY H-BOND REFINED ATOMS  (A):  6; 0.192; 0.500 REMARK 3  SYMMETRY H-BOND OTHERS   (A): NULL; NULL; NULL REMARK 3  SYMMETRY METAL-ION REFINED ATOMS (A): NULL; NULL; NULL REMARK 3  SYMMETRY METAL-ION OTHERS   (A): NULL; NULL; NULL REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS.  COUNT  RMS  WEIGHT REMARK 3  MAIN-CHAIN BOND REFINED ATOMS (A**2):  2884; 2.352; 2.000 REMARK 3  MAIN-CHAIN BOND OTHER ATOMS  (A**2):  896; 0.097; 2.000 REMARK 3  MAIN-CHAIN ANGLE REFINED ATOMS (A**2):  3571; 2.767; 2.500 REMARK 3  SIDE-CHAIN BOND REFINED ATOMS  (A**2):  1974; 4.582; 2.000 REMARK 3  SIDE-CHAIN ANGLE REFINED ATOMS (A**2):  1648; 5.849; 2.500 REMARK 3 REMARK 3 ANISOTROPIC THERMAL FACTOR RESTRAINTS.  COUNT  RMS  WEIGHT REMARK 3  RIGID-BOND RESTRAINTS   (A**2): NULL; NULL; NULL REMARK 3  SPHERICITY; FREE ATOMS   (A**2): NULL; NULL; NULL REMARK 3  SPHERICITY; BONDED ATOMS  (A**2): NULL; NULL; NULL REMARK 3 REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3  NUMBER OF DIFFERENT NCS GROUPS: NULL REMARK 3 REMARK 3 TLS DETAILS REMARK 3  NUMBER OF TLS GROUPS: 3 REMARK 3 REMARK 3 TLS GROUP: 1 REMARK 3  NUMBER OF COMPONENTS GROUP: 2 REMARK 3  COMPONENTS   C SSSEQI  TO C SSSEQI REMARK 3  RESIDUE RANGE:  A 1002   A  1011 REMARK 3  RESIDUE RANGE:  A 1062   A  1161 REMARK 3  ORIGIN FOR THE GROUP (A): −23.6840  58.4050  30.4850 REMARK 3  T TENSOR REMARK 3  T11: −0.2208 T22: −0.2598 REMARK 3  T33: −0.7033 T12:   0.0432 REMARK 3  T13: −0.0594 T23: −0.0241 REMARK 3  L TENSOR REMARK 3  L11:   3.1220 L22:   2.6914 REMARK 3  L33:   5.9787 L12:   0.8643 REMARK 3  L13:   0.6146 L23: −0.8632 REMARK 3  S TENSOR REMARK 3  S11: −0.1453 S12: −0.2225 S13:   0.2116 REMARK 3  S21:   0.0984 S22: −0.0656 S23: −0.0133 REMARK 3  S31: −0.1849 S32: −0.1761 S33:   0.2109 REMARK 3 REMARK 3  TLS GROUP: 2 REMARK 3  NUMBER OF COMPONENTS GROUP: 1 REMARK 3  COMPONENTS   C SSSEQI  TO C SSSEQI REMARK 3  RESIDUE RANGE:  A  1012   A  1061 REMARK 3  ORIGIN FOR THE GROUP (A): −35.0580  69.0010  11.9610 REMARK 3  T TENSOR REMARK 3  T11:   0.0414 T22: −0.0871 REMARK 3  T33: −0.4908 T12:   0.0577 REMARK 3  T13: −0.1559 T23: −0.0085 REMARK 3  L TENSOR REMARK 3  L11:   9.6406 L22:  16.6501 REMARK 3  L33:   7.1133 L12: −6.5961 REMARK 3  L13: −0.9803 L23:   3.2882 REMARK 3  S TENSOR REMARK 3  S11: −0.1628 S12: −0.1008 S13:   0.4114 REMARK 3  S21: −0.7585 S22: −0.1058 S23:   0.7355 REMARK 3  S31: −0.6590 S32: −0.6073 S33:   0.2686 REMARK 3 REMARK 3 TLS GROUP: 3 REMARK 3  NUMBER OF COMPONENTS GROUP: 2 REMARK 3  COMPONENTS   C SSSEQI  TO  C SSSEQI REMARK 3  RESIDUE RANGE:  A   29   A  230 REMARK 3  RESIDUE RANGE:  A  263  A  342 REMARK 3  ORIGIN FOR THE GROUP (A): −33.0740  20.0130  7.1220 REMARK 3  T TENSOR REMARK 3  T11: −0.0103 T22: −0.2341 REMARK 3  T33: −0.5401 T12: −0.0025 REMARK 3  T13: −0.0974 T23: −0.0034 REMARK 3  L TENSOR REMARK 3  L11:   2.3670 L22:   6.1551 REMARK 3  L33:   1.9314 L12:   2.1068 REMARK 3  L13:   0.8591 L23:   0.7864 REMARK 3  S TENSOR REMARK 3  S11: −0.0346 S12:   0.0267 S13: −0.2068 REMARK 3  S21: −0.5009 S22:   0.0712 S23:   0.2388 REMARK 3  S31:   0.3208 S32:   0.0002 S33: −0.0366 REMARK 3 REMARK 3 BULK SOLVENT MODELLING. REMARK 3  METHOD USED: MASK REMARK 3  PARAMETERS FOR MASK CALCULATION REMARK 3  VDW PROBE RADIUS  : 1.40 REMARK 3  ION PROBE RADIUS  : 0.80 REMARK 3  SHRINKAGE RADIUS  : 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: HYDROGENS HAVE BEEN ADDED IN THE REMARK 3 RIDING POSITIONS. WATER #548 HAS STRONG DIFFERENCE DENSITY BUT REMARK 3 WEAK 2FO-FC DENSITY. REMARK 4 REMARK 4 2RH1 COMPLIES WITH FORMAT V. 3.1, 01-AUG-2007 REMARK 100 REMARK 100 THIS ENTRY HAS BEEN PROCESSED BY RCSB. REMARK 100 THE RCSB ID CODE IS RCSB044849. REMARK 200 REMARK 200 EXPERIMENTAL DETAILS REMARK 200  EXPERIMENT TYPE     : X-RAY DIFFRACTION REMARK 200  DATE OF DATA COLLECTION  : 22-JUN-2007; 18-JUL-2007 REMARK 200  TEMPERATURE    (KELVIN): 78; 78 REMARK 200  PH         : 6.75 REMARK 200  NUMBER OF CRYSTALS USED   : 27 REMARK 200 REMARK 200  SYNCHROTRON    (Y/N): Y; Y REMARK 200  RADIATION SOURCE    : APS; APS REMARK 200  BEAMLINE      : 23-ID-B; 23-ID-B REMARK 200  X-RAY GENERATOR MODEL   : NULL REMARK 200  MONOCHROMATIC OR LAUE  (M/L): M REMARK 200  WAVELENGTH OR RANGE   (A): 1.03321; 1.03321 REMARK 200  MONOCHROMATOR     : DOUBLE CRYSTAL REMARK 200  OPTICS       : MIRRORS; MIRRORS REMARK 200 REMARK 200  DETECTOR TYPE     : CCD; CCD REMARK 200  DETECTOR MANUFACTURER   : MARMOSAIC 300 MM CCD; REMARK 200          MARMOSAIC 300 MM CCD REMARK 200  INTENSITY-INTEGRATION SOFTWARE: XDS REMARK 200  DATA SCALING SOFTWARE   : XDS REMARK 200 REMARK 200  NUMBER OF UNIQUE REFLECTIONS  : 26506 REMARK 200  RESOLUTION RANGE HIGH  (A): 2.400 REMARK 200  RESOLUTION RANGE LOW  (A): 20.000 REMARK 200  REJECTION CRITERIA (SIGMA(I)): −3.000 REMARK 200 REMARK 200 OVERALL. REMARK 200  COMPLETENESS FOR RANGE  (%): 99.2 REMARK 200  DATA REDUNDANCY    : NULL REMARK 200  R MERGE     (I): 0.12700 REMARK 200  R SYM      (I): NULL REMARK 200  <I/SIGMA(I)> FOR THE DATA SET  : 9.6200 REMARK 200 REMARK 200 IN THE HIGHEST RESOLUTION SHELL. REMARK 200  HIGHEST RESOLUTION SHELL, RANGE HIGH (A): 2.40 REMARK 200  HIGHEST RESOLUTION SHELL, RANGE LOW  (A): 2.50 REMARK 200  COMPLETENESS FOR SHELL  (%): 99.1 REMARK 200  DATA REDUNDANCY IN SHELL  : NULL REMARK 200  R MERGE FOR SHELL    (I): 0.67800 REMARK 200  R SYM FOR SHELL    (I): NULL REMARK 200  <I/SIGMA(I)> FOR SHELL   : 2.200 REMARK 200 REMARK 200 DIFFRACTION PROTOCOL: SINGLE WAVELENGTH REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT REMARK 200 SOFTWARE USED: PHASER REMARK 200 STARTING MODEL: PDB ENTRIES 1U19, 2LZM REMARK 200 REMARK 200 REMARK: THIS STRUCTURE IS A PART OF THE ROADMAP/PSI COMMUNITY REMARK 200  OUTREACH PROGRAM, NOT A SPECIFIC PSI TARGET. REMARK 280 REMARK 280 CRYSTAL REMARK 280 SOLVENT CONTENT, VS (%): 59.98 REMARK 280 MATTHEWS COEFFICIENT, VM (ANGSTROMS**3/DA): 3.07 REMARK 280 REMARK 280 CRYSTALLIZATION CONDITIONS: 30-35% V/V PEG 400, 0.1-0.2 M REMARK 280  NA2SO4, 0.1 M BIS-TRIS PROPANE PH 6.5-7.0, 5-7% 1,4- REMARK 280  BUTANEDIOL, 8-10% CHOLESTEROL, 52-50% MONOOLEIN, PH 6.75, REMARK 280  LIPIDIC MESOPHASE, TEMPERATURE 293 K REMARK 290 REMARK 290 CRYSTALLOGRAPHIC SYMMETRY REMARK 290 SYMMETRY OPERATORS FOR SPACE GROUP: C 1 2 1 REMARK 290 REMARK 290   SYMOP  SYMMETRY REMARK 290  NNNMMM  OPERATOR REMARK 290   1555  X, Y, Z REMARK 290   2555  −X, Y, −Z REMARK 290   3555  ½ + X, ½ + Y, Z REMARK 290   4555  ½ − X, ½ + Y, −Z REMARK 290 REMARK 290  WHERE NNN -> OPERATOR NUMBER REMARK 290    MMM -> TRANSLATION VECTOR REMARK 290 REMARK 290 CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS REMARK 290 THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM REMARK 290 RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY REMARK 290 RELATED MOLECULES. REMARK 290  SMTRY1 1 1.000000 0.000000 0.000000 0.00000 REMARK 290  SMTRY2 1 0.000000 1.000000 0.000000 0.00000 REMARK 290  SMTRY3 1 0.000000 0.000000 1.000000 0.00000 REMARK 290  SMTRY1 2 −1.000000 0.000000 0.000000 0.00000 REMARK 290  SMTRY2 2 0.000000 1.000000 0.000000 0.00000 REMARK 290  SMTRY3 2 0.000000 0.000000 −1.000000 0.00000 REMARK 290  SMTRY1 3 1.000000 0.000000 0.000000 53.15900 REMARK 290  SMTRY2 3 0.000000 1.000000 0.000000 84.62000 REMARK 290  SMTRY3 3 0.000000 0.000000 1.000000 0.00000 REMARK 290  SMTRY1 4 −1.000000 0.000000 0.000000 53.15900 REMARK 290  SMTRY2 4 0.000000 1.000000 0.000000 84.62000 REMARK 290  SMTRY3 4 0.000000 0.000000 −1.000000 0.00000 REMARK 290 REMARK 290 REMARK: NULL REMARK 300 REMARK 300 BIOMOLECULE: 1 REMARK 300 SEE REMARK 350 FOR THE AUTHOR PROVIDED AND/OR PROGRAM REMARK 300 GENERATED ASSEMBLY INFORMATION FOR THE STRUCTURE IN REMARK 300 THIS ENTRY. THE REMARK MAY ALSO PROVIDE INFORMATION ON REMARK 300 BURIED SURFACE AREA. REMARK 300 REMARK 300 REMARK: AUTHORS STATE THAT THE BIOLOGICAL UNIT IS UNKNOWN REMARK 350 REMARK 350 COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWN REMARK 350 BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE REMARK 350 MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS REMARK 350 GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND REMARK 350 CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN. REMARK 350 REMARK 350 BIOMOLECULE: 1 REMARK 350 SOFTWARE DETERMINED QUATERNARY STRUCTURE: MONOMERIC REMARK 350 SOFTWARE USED: PISA REMARK 350 APPLY THE FOLLOWING TO CHAINS: A REMARK 350  BIOMT1 1 1.000000 0.000000 0.000000 0.00000 REMARK 350  BIOMT2 1 0.000000 1.000000 0.000000 0.00000 REMARK 350  BIOMT3 1 0.000000 0.000000 1.000000 0.00000 REMARK 375 REMARK 375 SPECIAL POSITION REMARK 375 THE FOLLOWING ATOMS ARE FOUND TO BE WITHIN 0.15 ANGSTROMS REMARK 375 OF A SYMMETRY RELATED ATOM AND ARE ASSUMED TO BE ON SPECIAL REMARK 375 POSITIONS. REMARK 375 REMARK 375 ATOM RES CSSEQI REMARK 375   HOH A 520  LIES ON A SPECIAL POSITION. REMARK 465 REMARK 465 MISSING RESIDUES REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE REMARK 465 EXPERIMENT. (M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN REMARK 465 IDENTIFIER; SSEQ = SEQUENCE NUMBER; I = INSERTION CODE.) REMARK 465 REMARK 465  M RES C SSEQI REMARK 465  ASP A −6 REMARK 465  TYR A −5 REMARK 465  LYS A −4 REMARK 465  ASP A −3 REMARK 465  ASP A −2 REMARK 465  ASP A −1 REMARK 465  ALA A 0 REMARK 465  MET A 1 REMARK 465  GLY A 2 REMARK 465  GLN A 3 REMARK 465  PRO A 4 REMARK 465  GLY A 5 REMARK 465  ASN A 6 REMARK 465  GLY A 7 REMARK 465  SER A 8 REMARK 465  ALA A 9 REMARK 465  PHE A 10 REMARK 465  LEU A 11 REMARK 465  LEU A 12 REMARK 465  ALA A 13 REMARK 465  PRO A 14 REMARK 465  ASN A 15 REMARK 465  ARG A 16 REMARK 465  SER A 17 REMARK 465  HIS A 18 REMARK 465  ALA A 19 REMARK 465  PRO A 20 REMARK 465  ASP A 21 REMARK 465  HIS A 22 REMARK 465  ASP A 23 REMARK 465  VAL A 24 REMARK 465  THR A 25 REMARK 465  GLN A 26 REMARK 465  GLN A 27 REMARK 465  ARG A 28 REMARK 465  ARG A 343 REMARK 465  ARG A 344 REMARK 465  SER A 345 REMARK 465  SER A 346 REMARK 465  LEU A 347 REMARK 465  LYS A 348 REMARK 465  ALA A 349 REMARK 465  TYR A 350 REMARK 465  GLY A 351 REMARK 465  ASN A 352 REMARK 465  GLY A 353 REMARK 465  TYR A 354 REMARK 465  SER A 355 REMARK 465  SER A 356 REMARK 465  ASN A 357 REMARK 465  GLY A 358 REMARK 465  ASN A 359 REMARK 465  THR A 360 REMARK 465  GLY A 361 REMARK 465  GLU A 362 REMARK 465  GLN A 363 REMARK 465  SER A 364 REMARK 465  GLY A 365 REMARK 470 REMARK 470 MISSING ATOM REMARK 470 THE FOLLOWING RESIDUES HAVE MISSING ATOMS(M = MODEL NUMBER; REMARK 470 RES = RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER; REMARK 470 I = INSERTION CODE): REMARK 470  M RES CSSEQI ATOMS REMARK 470  ASP A 29  CG  OD1  OD2 REMARK 500 REMARK 500 GEOMETRY AND STEREOCHEMISTRY REMARK 500 SUBTOPIC: CLOSE CONTACTS IN SAME ASYMMETRIC UNIT REMARK 500 REMARK 500 THE FOLLOWING ATOMS ARE IN CLOSE CONTACT. REMARK 500 REMARK 500  ATM1  RES C  SSEQI  ATM2  RES C  SSEQI REMARK 500  SG  CYS A  341   O2  PLM A  415    1.88 REMARK 500 REMARK 500 REMARK: NULL REMARK 500 REMARK 500 GEOMETRY AND STEREOCHEMISTRY REMARK 500 SUBTOPIC: TORSION ANGLES REMARK 500 REMARK 500 TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS: REMARK 500 (M = MODEL NUMBER; RES = RESIDUE NAME; C = CHAIN IDENTIFIER; REMARK 500 SSEQ = SEQUENCE NUMBER; I = INSERTION CODE). REMARK 500 REMARK 500 STANDARD TABLE: REMARK 500 FORMAT: (10X, I3, 1X, A3, 1X, A1, I4, A1, 4X, F7.2, 3X, F7.2) REMARK 500 REMARK 500 EXPECTED VALUES: GJ KLEYWEGT AND TA JONES (1996). PHI/PSI- REMARK 500 CHOLOGY: RAMACHANDRAN REVISITED. STRUCTURE 4, 1395-1400 REMARK 500 REMARK 500  M RES CSSEQI   PSI   PHI REMARK 500  VAL A 86   −64.27  −100.05 REMARK 500  TYR A 141   −22.19   73.12 REMARK 500 REMARK 500 REMARK: NULL REMARK 600 REMARK 600 HETEROGEN REMARK 600 THE PALMITIC ACID (PLM) AND ACETAMIDE (ACM) GROUPS ARE REMARK 600 COVALENTLY LINKED TO THEIR RESPECTIVE CYSTEINE RESIDUES. REMARK 610 REMARK 610 MISSING HETEROATOM REMARK 610 THE FOLLOWING RESIDUES HAVE MISSING ATOMS (M = MODEL NUMBER; REMARK 610 RES = RESIDUE NAME; C = CHAIN IDENTIFIER; SSEQ = SEQUENCE NUMBER; REMARK 610 I = INSERTION CODE): REMARK 610  M RES C SSEQI REMARK 610  12P A 416 REMARK 800 REMARK 800 SITE REMARK 800 SITE_IDENTIFIER: AC1 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE 12P A 416 REMARK 800 SITE_IDENTIFIER: AC2 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE ACM A 411 REMARK 800 SITE_IDENTIFIER: AC3 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE BU1 A 409 REMARK 800 SITE_IDENTIFIER: AC5 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CAU A 408 REMARK 800 SITE_IDENTIFIER: AC6 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CLR A 412 REMARK 800 SITE_IDENTIFIER: AC7 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE CLR A 413 REMARK 800 SITE_IDENTIFIER: AC9 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE MAL A 401 REMARK 800 SITE_IDENTIFIER: BC1 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE PLM A 415 REMARK 800 SITE_IDENTIFIER: BC2 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 402 REMARK 800 SITE_IDENTIFIER: BC3 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 403 REMARK 800 SITE_IDENTIFIER: BC4 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 404 REMARK 800 SITE_IDENTIFIER: BC5 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 405 REMARK 800 SITE_IDENTIFIER: BC6 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 406 REMARK 800 SITE_IDENTIFIER: BC7 REMARK 800 SITE_DESCRIPTION: BINDING SITE FOR RESIDUE SO4 A 407 REMARK 999 REMARK 999 SEQUENCE THE STRUCTURE IS AN INTERNAL FUSION PROTEIN WITH REMARK 999 LYSOZYME. AN OFFSET 1000 HAS BEEN ADDED TO ORIGINAL REMARK 999 SEQUENCE DATABASE RESIDUE NUMBERS (2-161) OF THE LYSOZYME REMARK 999 PART IN COORDINATES TO DISTINGUISH THE LYSOZYME PART IN THE REMARK 999 CHAIN. THEREFORE THE RESIDUES OF LYSOZYME PART HAVE NUMBERS REMARK 999 A1002-A1161. DBREF 2RH1 A  1  230  UNP  P07550  ADRB2_HUMAN   1  230 DBREF 2RH1 A 1002  1161  UNP  P00720  LYS_BPT4   2  161 DBREF 2RH1 A 263   365  UNP  P07550  ADRB2_HUMAN  263  365 SEQADV 2RH1 ASP A  −6 UNP P07550    EXPRESSION TAG SEQADV 2RH1 TYR A  −5 UNP P07550    EXPRESSION TAG SEQADV 2RH1 LYS A  −4 UNP P07550    EXPRESSION TAG SEQADV 2RH1 ASP A  −3 UNP P07550    EXPRESSION TAG SEQADV 2RH1 ASP A  −2 UNP P07550    EXPRESSION TAG SEQADV 2RH1 ASP A  −1 UNP P07550    EXPRESSION TAG SEQADV 2RH1 ALA A  0 UNP P07550    EXPRESSION TAG SEQADV 2RH1 GLU A 187 UNP P07550  ASN  187 ENGINEERED SEQADV 2RH1 THR A 1054 UNP P00720  CYS  54 ENGINEERED SEQADV 2RH1 ALA A 1097 UNP P00720  CYS  97 ENGINEERED SEQRES 1 A 500 ASP TYR LYS ASP ASP ASP ALA MET GLY GLN PRO GLY ASN SEQRES 2 A 500 GLY SER ALA PHE LEU LEU ALA PRO ASN ARG SER HIS ALA SEQRES 3 A 500 PRO ASP HIS ASP VAL THR GLN GLN ARG ASP GLU VAL TRP SEQRES 4 A 500 VAL VAL GLY MET GLY ILE VAL MET SER LEU ILE VAL LEU SEQRES 5 A 500 ALA ILE VAL PHE GLY ASN VAL LEU VAL ILE THR ALA ILE SEQRES 6 A 500 ALA LYS PHE GLU ARG LEU GLN THR VAL THR ASN TYR PHE SEQRES 7 A 500 ILE THR SER LEU ALA CYS ALA ASP LEU VAL MET GLY LEU SEQRES 8 A 500 ALA VAL VAL PRO PHE GLY ALA ALA HIS ILE LEU MET LYS SEQRES 9 A 500 MET TRP THR PHE GLY ASN PHE TRP CYS GLU PHE TRP THR SEQRES 10 A 500 SER ILE ASP VAL LEU CYS VAL THR ALA SER ILE GLU THR SEQRES 11 A 500 LEU CYS VAL ILE ALA VAL ASP ARG TYR PHE ALA ILE THR SEQRES 12 A 500 SER PRO PHE LYS TYR GLN SER LEU LEU THR LYS ASN LYS SEQRES 13 A 500 ALA ARG VAL ILE ILE LEU MET VAL TRP ILE VAL SER GLY SEQRES 14 A 500 LEU THR SER PHE LEU PRO ILE GLN MET HIS TRP TYR ARG SEQRES 15 A 500 ALA THR HIS GLN GLU ALA ILE ASN CYS TYR ALA GLU GLU SEQRES 16 A 500 THR CYS CYS ASP PHE PHE THR ASN GLN ALA TYR ALA ILE SEQRES 17 A 500 ALA SER SER ILE VAL SER PHE TYR VAL PRO LEU VAL ILE SEQRES 18 A 500 MET VAL PHE VAL TYR SER ARG VAL PHE GLN GLU ALA LYS SEQRES 19 A 500 ARG GLN LEU ASN ILE PHE GLU MET LEU ARG ILE ASP GLU SEQRES 20 A 500 GLY LEU ARG LEU LYS ILE TYR LYS ASP THR GLU GLY TYR SEQRES 21 A 500 TYR THR ILE GLY ILE GLY HIS LEU LEU THR LYS SER PRO SEQRES 22 A 500 SER LEU ASN ALA ALA LYS SER GLU LEU ASP LYS ALA ILE SEQRES 23 A 500 GLY ARG ASN THR ASN GLY VAL ILE THR LYS ASP GLU ALA SEQRES 24 A 500 GLU LYS LEU PHE ASN GLN ASP VAL ASP ALA ALA VAL ARG SEQRES 25 A 500 GLY ILE LEU ARG ASN ALA LYS LEU LYS PRO VAL TYR ASP SEQRES 26 A 500 SER LEU ASP ALA VAL ARG ARG ALA ALA LEU ILE ASN MET SEQRES 27 A 500 VAL PHE GLN MET GLY GLU THR GLY VAL ALA GLY PHE THR SEQRES 28 A 500 ASN SER LEU ARG MET LEU GLN GLN LYS ARG TRP ASP GLU SEQRES 29 A 500 ALA ALA VAL ASN LEU ALA LYS SER ARG TRP TYR ASN GLN SEQRES 30 A 500 THR PRO ASN ARG ALA LYS ARG VAL ILE THR THR PHE ARG SEQRES 31 A 500 THR GLY THR TRP ASP ALA TYR LYS PHE CYS LEU LYS GLU SEQRES 32 A 500 HIS LYS ALA LEU LYS THR LEU GLY ILE ILE MET GLY THR SEQRES 33 A 500 PHE THR LEU CYS TRP LEU PRO PHE PHE ILE VAL ASN ILE SEQRES 34 A 500 VAL HIS VAL ILE GLN ASP ASN LEU ILE ARG LYS GLU VAL SEQRES 35 A 500 TYR ILE LEU LEU ASN TRP ILE GLY TYR VAL ASN SER GLY SEQRES 36 A 500 PHE ASN PRO LEU ILE TYR CYS ARG SER PRO ASP PHE ARG SEQRES 37 A 500 ILE ALA PHE GLN GLU LEU LEU CYS LEU ARG ARG SER SER SEQRES 38 A 500 LEU LYS ALA TYR GLY ASN GLY TYR SER SER ASN GLY ASN SEQRES 39 A 500 THR GLY GLU GLN SER GLY HET MAL A 401 23 HET SO4 A 402 5 HET SO4 A 403 5 HET SO4 A 404 5 HET SO4 A 405 5 HET SO4 A 406 5 HET SO4 A 407 5 HET CAU A 408 22 HET BU1 A 409 6 HET BU1 A 410 6 HET ACM A 411 4 HET CLR A 412 28 HET CLR A 413 28 HET CLR A 414 28 HET PLM A 415 17 HET 12P A 416 21 HETNAM  MAL MALTOSE HETNAM  SO4 SULFATE ION HETNAM  CAU (2S)-1-(9H-CARBAZOL-4-YLOXY)-3-(ISOPROPYLAMINO)PROPAN- HETNAM 2 CAU 2-OL HETNAM  BU1 1,4-BUTANEDIOL HETNAM  ACM ACETAMIDE HETNAM  CLR CHOLESTEROL HETNAM  PLM PALMITIC ACID HETNAM  12P DODECAETHYLENE GLYCOL HETSYN  CAU (S)-CARAZOLOL HETSYN  12P POLYETHYLENE GLYCOL PEG400 FORMUL  2 MAL C12 H22 O11 FORMUL  3 SO4 6(O4 S 2-) FORMUL  9 CAU C18 H22 N2 O2 FORMUL 10 BU1 2(C4 H10 O2) FORMUL 12 ACM C2 H5 N O FORMUL 13 CLR 3(C27 H46 O) FORMUL 16 PLM C16 H32 O2 FORMUL 17 12P C24 H50 O13 FORMUL 18 HOH *48(H2 O) HELIX 1 1 ASP A 29 LYS A 60 1 32 HELIX 2 2 VAL A 67 MET A 96 1 30 HELIX 3 3 ASN A 103 THR A 136 1 34 HELIX 4 4 LYS A 147 MET A 171 1 25 HELIX 5 5 HIS A 178 GLU A 187 1 10 HELIX 6 6 GLN A 197 GLN A 229 1 33 HELIX 7 7 LYS A 267 ILE A 298 1 32 HELIX 8 8 LYS A 305 ARG A 328 1 24 HELIX 9 9 PRO A 330 CYS A 341 1 12 HELIX 10 10 ILE A 1003 GLU A 1011 1 9 HELIX 11 11 LEU A 1039 ILE A 1050 1 12 HELIX 12 12 LYS A 1060 ARG A 1080 1 21 HELIX 13 13 ALA A 1082 SER A 1090 1 9 HELIX 14 14 ALA A 1093 MET A 1106 1 14 HELIX 15 15 GLU A 1108 GLY A 1113 1 6 HELIX 16 16 THR A 1115 GLN A 1123 1 9 HELIX 17 17 TRP A 1126 ALA A 1134 1 9 HELIX 18 18 ARG A 1137 GLN A 1141 1 5 HELIX 19 19 PRO A 1143 THR A 1155 1 13 SHEET 1 1 4 LYS A 1016 ASP A 1020 0 SHEET 2 1 4 TYR A 1024 GLY A 1028 0 SHEET 3 1 4 HIS A 1031 THR A 1034 0 SHEET 4 1 4 GLY A 1056 THR A 1059 0 SSBOND  1 CYS A  106  CYS A  191  1555   1555   2.05 SSBOND  2 CYS A  184  CYS A  190  1555   1555   2.06 LINK  SG CYS A 265     C2 ACM A 411  1555   1555   1.61 LINK  SG CYS A 341     C1 PLM A 415 1555  1555  1.62 SITE 1 AC1 2 ASP A 1072 HOH A 538 SITE 1 AC2 1 CYS A 265 SITE 1 AC3 4 LYS A 263 PHE A 264 HIS A 269 HOH A 502 SITE 1 AC5 4 ASP A 113 PHE A 193 ASN A 312 TYR A 316 SITE 1 AC6 1 ILE A 112 SITE 1 AC7 1 HOH A 520 SITE 1 AC9 7 GLU A 1011 GLY A 1030 LEU A 1032 ASP A 1070 SITE 2 AC9 7 VAL A 1103 PHE A 1104 ARG A 1145 SITE 1 BC1 2 LEU A 339 CYS A 341 SITE 1 BC2 5 VAL A 67 THR A 68 ARG A 131 TYR A 141 SITE 2 BC2 5 SER A 143 SITE 1 BC3 4 PHE A 264 LYS A 270 LYS A 273 ARG A 328 SITE 1 BC4 5 PHE A 1114 THR A 1115 ASN A 1116 SER A 1117 SITE 2 BC4 5 ASN A 1132 SITE 1 BC5 6 PRO A 1143 ASN A 1144 ARG A 1145 HOH A 512 SITE 2 BC5 6 HOH A 526 HOH A 531 SITE 1 BC6 1 ARG A 1095 SITE 1 BC7 2 LEU A 1015 LYS A 1016 CRYST1 106.318 169.240 40.154 90.00 105.62 90.00 C 1 2 1   4 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE1 0.009406 0.000000 0.002630 0.00000 SCALE2 0.000000 0.005909 0.000000 0.00000 SCALE3 0.000000 0.000000 0.025859 0.00000 ATOM 1 N ASP A 29 −52.822 −1.611 23.137 1.00 98.48 N ATOM 2 CA ASP A 29 −51.922 −2.262 22.148 1.00 98.06 C ATOM 3 C ASP A 29 −52.178 −1.713 20.742 1.00 97.74 C ATOM 4 O ASP A 29 −51.291 −1.100 20.143 1.00 96.54 O ATOM 5 CB ASP A 29 −52.106 −3.786 22.184 1.00 97.64 C ATOM 6 N GLU A 30 −53.394 −1.944 20.236 1.00 98.37 N ATOM 7 CA GLU A 30 −53.821 −1.515 18.887 1.00 98.17 C ATOM 8 C GLU A 30 −54.424 −0.104 18.879 1.00 98.57 C ATOM 9 O GLU A 30 −54.197 0.649 17.943 1.00 99.79 O ATOM 10 CB GLU A 30 −54.840 −2.498 18.305 1.00 99.00 C ATOM 11 CG GLU A 30 −54.377 −3.969 18.286 1.00 99.46 C ATOM 12 CD GLU A 30 −55.432 −4.928 17.733 1.00 98.77 C ATOM 13 OE1 GLU A 30 −56.228 −4.527 16.853 1.00 97.60 O ATOM 14 OE2 GLU A 30 −55.463 −6.092 18.185 1.00 99.75 O ATOM 15 N VAL A 31 −55.190 0.248 19.918 1.00 97.89 N ATOM 16 CA VAL A 31 −55.757 1.618 20.079 1.00 96.48 C ATOM 17 C VAL A 31 −54.643 2.678 20.185 1.00 95.12 C ATOM 18 O VAL A 31 −54.838 3.837 19.803 1.00 93.37 O ATOM 19 CB VAL A 31 −56.729 1.697 21.306 1.00 96.71 C ATOM 20 CG1 VAL A 31 −57.185 3.135 21.580 1.00 96.80 C ATOM 21 CG2 VAL A 31 −57.948 0.806 21.077 1.00 96.80 C ATOM 22 N TRP A 32 −53.486 2.259 20.709 1.00 95.21 N ATOM 23 CA TRP A 32 −52.267 3.063 20.735 1.00 94.60 C ATOM 24 C TRP A 32 −51.837 3.491 19.319 1.00 93.60 C ATOM 25 O TRP A 32 −51.335 4.603 19.140 1.00 91.48 O ATOM 26 CB TRP A 32 −51.129 2.273 21.427 1.00 96.93 C ATOM 27 CG TRP A 32 −49.770 2.899 21.304 1.00 98.27 C ATOM 28 CD1 TRP A 32 −49.169 3.734 22.197 1.00 100.48 C ATOM 29 CD2 TRP A 32 −48.844 2.737 20.216 1.00 100.89 C ATOM 30 NE1 TRP A 32 −47.924 4.105 21.737 1.00 101.70 N ATOM 31 CE2 TRP A 32 −47.700 3.507 20.523 1.00 102.25 C ATOM 32 CE3 TRP A 32 −48.874 2.013 19.010 1.00 100.84 C ATOM 33 CZ2 TRP A 32 −46.587 3.578 19.667 1.00 101.34 C ATOM 34 CZ3 TRP A 32 −47.771 2.082 18.158 1.00 100.68 C ATOM 35 CH2 TRP A 32 −46.640 2.860 18.493 1.00 101.20 C ATOM 36 N VAL A 33 −52.036 2.600 18.332 1.00 92.39 N ATOM 37 CA VAL A 33 −51.667 2.852 16.922 1.00 91.10 C ATOM 38 C VAL A 33 −52.435 4.017 16.332 1.00 89.89 C ATOM 39 O VAL A 33 −51.827 4.918 15.748 1.00 88.01 O ATOM 40 CB VAL A 33 −51.920 1.621 16.012 1.00 91.28 C ATOM 41 CG1 VAL A 33 −51.625 1.950 14.553 1.00 93.68 C ATOM 42 CG2 VAL A 33 −51.080 0.443 16.467 1.00 93.38 C ATOM 43 N VAL A 34 −53.762 3.986 16.490 1.00 88.20 N ATOM 44 CA VAL A 34 −54.638 5.078 16.041 1.00 87.62 C ATOM 45 C VAL A 34 −54.145 6.388 16.649 1.00 87.34 C ATOM 46 O VAL A 34 −53.871 7.334 15.921 1.00 88.42 O ATOM 47 CB VAL A 34 −56.126 4.852 16.422 1.00 87.61 C ATOM 48 CG1 VAL A 34 −56.972 6.064 16.046 1.00 87.66 C ATOM 49 CG2 VAL A 34 −56.684 3.592 15.755 1.00 85.12 C ATOM 50 N GLY A 35 −54.036 6.413 17.981 1.00 86.61 N ATOM 51 CA GLY A 35 −53.524 7.576 18.738 1.00 86.08 C ATOM 52 C GLY A 35 −52.123 8.048 18.358 1.00 85.30 C ATOM 53 O GLY A 35 −51.837 9.245 18.408 1.00 85.51 O ATOM 54 N MET A 36 −51.257 7.110 17.981 1.00 85.55 N ATOM 55 CA MET A 36 −49.919 7.431 17.486 1.00 84.86 C ATOM 56 C MET A 36 −50.004 7.888 16.029 1.00 85.06 C ATOM 57 O MET A 36 −49.216 8.733 15.590 1.00 86.04 O ATOM 58 CB MET A 36 −49.001 6.214 17.596 1.00 86.69 C ATOM 59 CG MET A 36 −47.509 6.496 17.400 1.00 88.99 C ATOM 60 SD MET A 36 −46.800 7.642 18.613 1.00 102.42 S ATOM 61 CE MET A 36 −47.213 6.857 20.179 1.00 100.39 C ATOM 62 N GLY A 37 −50.959 7.324 15.287 1.00 83.06 N ATOM 63 CA GLY A 37 −51.227 7.713 13.911 1.00 82.58 C ATOM 64 C GLY A 37 −51.710 9.144 13.811 1.00 81.74 C ATOM 65 O GLY A 37 −51.171 9.912 13.024 1.00 83.08 O ATOM 66 N ILE A 38 −52.724 9.494 14.611 1.00 81.34 N ATOM 67 CA ILE A 38 −53.260 10.869 14.659 1.00 80.88 C ATOM 68 C ILE A 38 −52.153 11.874 15.003 1.00 80.13 C ATOM 69 O ILE A 38 −52.121 12.964 14.453 1.00 81.84 O ATOM 70 CB ILE A 38 −54.432 11.032 15.686 1.00 81.07 C ATOM 71 CG1 ILE A 38 −55.635 10.132 15.356 1.00 81.29 C ATOM 72 CG2 ILE A 38 −54.912 12.479 15.745 1.00 80.45 C ATOM 73 CD1 ILE A 38 −56.310 10.413 14.026 1.00 85.05 C ATOM 74 N VAL A 39 −51.255 11.496 15.911 1.00 79.45 N ATOM 75 CA VAL A 39 −50.128 12.346 16.306 1.00 79.67 C ATOM 76 C VAL A 39 −49.166 12.575 15.154 1.00 79.83 C ATOM 77 O VAL A 39 −48.803 13.717 14.863 1.00 81.09 O ATOM 78 CB VAL A 39 −49.352 11.740 17.496 1.00 81.11 C ATOM 79 CG1 VAL A 39 −47.996 12.425 17.671 1.00 78.01 C ATOM 80 CG2 VAL A 39 −50.185 11.843 18.770 1.00 81.49 C ATOM 81 N MET A 40 −48.754 11.492 14.506 1.00 80.50 N ATOM 82 CA MET A 40 −47.881 11.589 13.320 1.00 80.67 C ATOM 83 C MET A 40 −48.575 12.333 12.155 1.00 80.62 C ATOM 84 O MET A 40 −47.915 13.033 11.390 1.00 78.80 O ATOM 85 CB MET A 40 −47.405 10.197 12.870 1.00 80.79 C ATOM 86 CG MET A 40 −46.294 9.613 13.745 1.00 80.56 C ATOM 87 SD MET A 40 −45.995 7.863 13.419 1.00 83.92 S ATOM 88 CE MET A 40 −44.486 7.588 14.344 1.00 83.67 C ATOM 89 N SER A 41 −49.896 12.177 12.034 1.00 79.41 N ATOM 90 CA SER A 41 −50.672 12.898 11.020 1.00 79.83 C ATOM 91 C SER A 41 −50.651 14.403 11.269 1.00 80.21 C ATOM 92 O SER A 41 −50.521 15.181 10.324 1.00 82.42 O ATOM 93 CB SER A 41 −52.104 12.394 10.976 1.00 79.47 C ATOM 94 OG SER A 41 −52.128 11.044 10.561 1.00 82.68 O ATOM 95 N LEU A 42 −50.778 14.805 12.534 1.00 79.85 N ATOM 96 CA LEU A 42 −50.675 16.219 12.908 1.00 79.83 C ATOM 97 C LEU A 42 −49.256 16.752 12.687 1.00 78.29 C ATOM 98 O LEU A 42 −49.093 17.917 12.355 1.00 80.15 O ATOM 99 CB LEU A 42 −51.106 16.448 14.364 1.00 80.71 C ATOM 100 CG LEU A 42 −52.586 16.242 14.714 1.00 81.76 C ATOM 101 CD1 LEU A 42 −52.774 16.400 16.213 1.00 83.86 C ATOM 102 CD2 LEU A 42 −53.508 17.191 13.956 1.00 82.80 C ATOM 103 N ILE A 43 −48.243 15.902 12.876 1.00 77.38 N ATOM 104 CA ILE A 43 −46.850 16.274 12.593 1.00 77.38 C ATOM 105 C ILE A 43 −46.683 16.604 11.112 1.00 77.46 C ATOM 106 O ILE A 43 −46.072 17.608 10.771 1.00 77.58 O ATOM 107 CB ILE A 43 −45.845 15.155 12.995 1.00 78.54 C ATOM 108 CG1 ILE A 43 −45.698 15.080 14.520 1.00 80.06 C ATOM 109 CG2 ILE A 43 −44.473 15.387 12.366 1.00 74.64 C ATOM 110 CD1 ILE A 43 −44.923 13.845 15.002 1.00 77.13 C ATOM 111 N VAL A 44 −47.229 15.750 10.247 1.00 77.58 N ATOM 112 CA VAL A 44 −47.195 15.967 8.801 1.00 76.60 C ATOM 113 C VAL A 44 −47.951 17.251 8.416 1.00 78.14 C ATOM 114 O VAL A 44 −47.445 18.053 7.629 1.00 76.84 O ATOM 115 CB VAL A 44 −47.769 14.755 8.041 1.00 76.85 C ATOM 116 CG1 VAL A 44 −47.967 15.079 6.567 1.00 75.75 C ATOM 117 CG2 VAL A 44 −46.856 13.533 8.219 1.00 70.47 C ATOM 118 N LEU A 45 −49.150 17.434 8.972 1.00 78.66 N ATOM 119 CA LEU A 45 −49.954 18.638 8.719 1.00 78.89 C ATOM 120 C LEU A 45 −49.219 19.901 9.163 1.00 79.68 C ATOM 121 O LEU A 45 −49.246 20.900 8.460 1.00 80.46 O ATOM 122 CB LEU A 45 −51.306 18.555 9.433 1.00 80.42 C ATOM 123 CG LEU A 45 −52.374 19.590 9.062 1.00 83.12 C ATOM 124 CD1 LEU A 45 −52.835 19.414 7.616 1.00 86.47 C ATOM 125 CD2 LEU A 45 −53.555 19.485 10.008 1.00 82.85 C ATOM 126 N ALA A 46 −48.571 19.830 10.330 1.00 78.89 N ATOM 127 CA ALA A 46 −47.783 20.933 10.893 1.00 76.32 C ATOM 128 C ALA A 46 −46.639 21.352 9.985 1.00 75.76 C ATOM 129 O ALA A 46 −46.453 22.539 9.731 1.00 78.99 O ATOM 130 CB ALA A 46 −47.227 20.535 12.244 1.00 75.15 C ATOM 131 N ILE A 47 −45.885 20.371 9.505 1.00 72.82 N ATOM 132 CA ILE A 47 −44.751 20.613 8.596 1.00 72.71 C ATOM 133 C ILE A 47 −45.206 21.216 7.267 1.00 69.65 C ATOM 134 O ILE A 47 −44.651 22.209 6.815 1.00 68.44 O ATOM 135 CB ILE A 47 −43.984 19.310 8.290 1.00 73.57 C ATOM 136 CG1 ILE A 47 −43.276 18.777 9.540 1.00 73.61 C ATOM 137 CG2 ILE A 47 −42.961 19.534 7.179 1.00 73.63 C ATOM 138 CD1 ILE A 47 −42.811 17.358 9.399 1.00 71.65 C ATOM 139 N VAL A 48 −46.216 20.606 6.659 1.00 68.32 N ATOM 140 CA VAL A 48 −46.721 21.051 5.360 1.00 69.33 C ATOM 141 C VAL A 48 −47.390 22.440 5.463 1.00 71.00 C ATOM 142 O VAL A 48 −47.115 23.305 4.638 1.00 72.48 O ATOM 143 CB VAL A 48 −47.692 20.011 4.722 1.00 69.55 C ATOM 144 CG1 VAL A 48 −48.178 20.489 3.364 1.00 64.85 C ATOM 145 CG2 VAL A 48 −47.008 18.642 4.585 1.00 66.28 C ATOM 146 N PHE A 49 −48.248 22.640 6.468 1.00 70.84 N ATOM 147 CA PHE A 49 −48.951 23.916 6.654 1.00 69.45 C ATOM 148 C PHE A 49 −47.994 25.077 6.803 1.00 71.34 C ATOM 149 O PHE A 49 −48.039 26.025 6.017 1.00 71.00 O ATOM 150 CB PHE A 49 −49.869 23.866 7.886 1.00 70.88 C ATOM 151 CG PHE A 49 −50.669 25.123 8.103 1.00 71.45 C ATOM 152 CD1 PHE A 49 −51.932 25.254 7.554 1.00 72.92 C ATOM 153 CD2 PHE A 49 −50.161 26.176 8.855 1.00 75.73 C ATOM 154 CE1 PHE A 49 −52.678 26.414 7.751 1.00 73.41 C ATOM 155 CE2 PHE A 49 −50.905 27.336 9.052 1.00 74.53 C ATOM 156 CZ PHE A 49 −52.162 27.450 8.498 1.00 69.47 C ATOM 157 N GLY A 50 −47.133 24.985 7.821 1.00 71.43 N ATOM 158 CA GLY A 50 −46.193 26.040 8.158 1.00 67.82 C ATOM 159 C GLY A 50 −45.210 26.409 7.066 1.00 68.17 C ATOM 160 O GLY A 50 −44.952 27.592 6.852 1.00 67.71 O ATOM 161 N ASN A 51 −44.662 25.402 6.381 1.00 66.99 N ATOM 162 CA ASN A 51 −43.713 25.636 5.273 1.00 67.98 C ATOM 163 C ASN A 51 −44.414 26.145 3.998 1.00 71.16 C ATOM 164 O ASN A 51 −43.847 26.968 3.274 1.00 69.41 O ATOM 165 CB ASN A 51 −42.890 24.395 4.997 1.00 68.88 C ATOM 166 CG ASN A 51 −41.927 24.083 6.134 1.00 71.66 C ATOM 167 OD1 ASN A 51 −40.914 24.758 6.305 1.00 69.08 O ATOM 168 ND2 ASN A 51 −42.241 23.063 6.913 1.00 68.08 N ATOM 169 N VAL A 52 −45.631 25.656 3.730 1.00 67.56 N ATOM 170 CA VAL A 52 −46.453 26.245 2.687 1.00 70.68 C ATOM 171 C VAL A 52 −46.616 27.731 2.994 1.00 71.64 C ATOM 172 O VAL A 52 −46.463 28.558 2.108 1.00 72.88 O ATOM 173 CB VAL A 52 −47.860 25.599 2.575 1.00 69.27 C ATOM 174 CG1 VAL A 52 −48.837 26.549 1.903 1.00 71.90 C ATOM 175 CG2 VAL A 52 −47.784 24.286 1.829 1.00 67.05 C ATOM 176 N LEU A 53 −46.921 28.049 4.256 1.00 73.15 N ATOM 177 CA LEU A 53 −47.156 29.441 4.688 1.00 72.57 C ATOM 178 C LEU A 53 −45.913 30.335 4.500 1.00 73.72 C ATOM 179 O LEU A 53 −46.043 31.494 4.120 1.00 76.55 O ATOM 180 CB LEU A 53 −47.632 29.482 6.147 1.00 72.34 C ATOM 181 CG LEU A 53 −48.422 30.705 6.593 1.00 74.13 C ATOM 182 CD1 LEU A 53 −49.812 30.716 5.973 1.00 73.24 C ATOM 183 CD2 LEU A 53 −48.530 30.738 8.109 1.00 76.12 C ATOM 184 N VAL A 54 −44.723 29.789 4.765 1.00 72.68 N ATOM 185 CA VAL A 54 −43.458 30.517 4.563 1.00 70.92 C ATOM 186 C VAL A 54 −43.183 30.795 3.076 1.00 72.46 C ATOM 187 O VAL A 54 −42.786 31.903 2.711 1.00 72.67 O ATOM 188 CB VAL A 54 −42.255 29.741 5.161 1.00 73.28 C ATOM 189 CG1 VAL A 54 −40.937 30.380 4.769 1.00 72.16 C ATOM 190 CG2 VAL A 54 −42.376 29.647 6.682 1.00 69.63 C ATOM 191 N ILE A 55 −43.395 29.787 2.233 1.00 69.98 N ATOM 192 CA ILE A 55 −43.164 29.906 0.791 1.00 68.58 C ATOM 193 C ILE A 55 −44.076 30.960 0.177 1.00 69.05 C ATOM 194 O ILE A 55 −43.606 31.844 −0.552 1.00 65.40 O ATOM 195 CB ILE A 55 −43.379 28.569 0.090 1.00 68.25 C ATOM 196 CG1 ILE A 55 −42.253 27.591 0.454 1.00 71.45 C ATOM 197 CG2 ILE A 55 −43.430 28.766 −1.409 1.00 72.11 C ATOM 198 CD1 ILE A 55 −42.573 26.152 0.127 1.00 71.51 C ATOM 199 N THR A 56 −45.369 30.840 0.491 1.00 70.28 N ATOM 200 CA THR A 56 −46.403 31.770 0.067 1.00 69.62 C ATOM 201 C THR A 56 −46.078 33.211 0.454 1.00 71.19 C ATOM 202 O THR A 56 −46.188 34.089 −0.372 1.00 72.89 O ATOM 203 CB THR A 56 −47.780 31.399 0.686 1.00 71.36 C ATOM 204 OG1 THR A 56 −48.082 30.019 0.434 1.00 65.43 O ATOM 205 CG2 THR A 56 −48.896 32.281 0.110 1.00 69.44 C ATOM 206 N ALA A 57 −45.678 33.423 1.714 1.00 71.45 N ATOM 207 CA ALA A 57 −45.368 34.762 2.263 1.00 70.84 C ATOM 208 C ALA A 57 −44.180 35.464 1.584 1.00 71.90 C ATOM 209 O ALA A 57 −44.220 36.665 1.339 1.00 70.99 O ATOM 210 CB ALA A 57 −45.119 34.666 3.767 1.00 67.19 C ATOM 211 N ILE A 58 −43.128 34.712 1.289 1.00 71.62 N ATOM 212 CA ILE A 58 −41.960 35.250 0.586 1.00 71.02 C ATOM 213 C ILE A 58 −42.294 35.462 −0.910 1.00 76.54 C ATOM 214 O ILE A 58 −41.898 36.471 −1.503 1.00 79.19 O ATOM 215 CB ILE A 58 −40.758 34.321 0.745 1.00 69.83 C ATOM 216 CG1 ILE A 58 −40.343 34.235 2.227 1.00 69.16 C ATOM 217 CG2 ILE A 58 −39.585 34.805 −0.082 1.00 69.14 C ATOM 218 CD1 ILE A 58 −39.371 33.129 2.513 1.00 70.35 C ATOM 219 N ALA A 59 −43.016 34.511 −1.505 1.00 74.84 N ATOM 220 CA ALA A 59 −43.420 34.599 −2.913 1.00 75.72 C ATOM 221 C ALA A 59 −44.422 35.722 −3.174 1.00 76.56 C ATOM 222 O ALA A 59 −44.416 36.311 −4.256 1.00 77.62 O ATOM 223 CB ALA A 59 −43.999 33.268 −3.385 1.00 72.21 C ATOM 224 N LYS A 60 −45.271 36.012 −2.184 1.00 76.98 N ATOM 225 CA LYS A 60 −46.336 37.010 −2.323 1.00 76.24 C ATOM 226 C LYS A 60 −45.932 38.438 −1.965 1.00 77.94 C ATOM 227 O LYS A 60 −46.282 39.371 −2.694 1.00 75.40 O ATOM 228 CB LYS A 60 −47.532 36.605 −1.466 1.00 75.25 C ATOM 229 CG LYS A 60 −48.667 37.578 −1.530 1.00 77.75 C ATOM 230 CD LYS A 60 −49.959 36.950 −1.164 1.00 78.52 C ATOM 231 CE LYS A 60 −51.028 37.983 −1.189 1.00 79.74 C ATOM 232 NZ LYS A 60 −52.299 37.333 −1.081 1.00 83.19 N ATOM 233 N PHE A 61 −45.209 38.597 −0.856 1.00 78.18 N ATOM 234 CA PHE A 61 −44.861 39.919 −0.334 1.00 79.67 C ATOM 235 C PHE A 61 −43.457 40.379 −0.743 1.00 81.98 C ATOM 236 O PHE A 61 −42.443 39.855 −0.253 1.00 78.47 O ATOM 237 CB PHE A 61 −45.055 39.943 1.185 1.00 78.50 C ATOM 238 CG PHE A 61 −46.488 39.744 1.589 1.00 77.03 C ATOM 239 CD1 PHE A 61 −46.946 38.513 2.013 1.00 74.37 C ATOM 240 CD2 PHE A 61 −47.385 40.799 1.536 1.00 79.08 C ATOM 241 CE1 PHE A 61 −48.267 38.334 2.380 1.00 77.21 C ATOM 242 CE2 PHE A 61 −48.708 40.621 1.903 1.00 77.13 C ATOM 243 CZ PHE A 61 −49.141 39.382 2.326 1.00 75.36 C ATOM 244 N GLU A 62 −43.450 41.369 −1.648 1.00 84.43 N ATOM 245 CA GLU A 62 −42.247 42.031 −2.208 1.00 86.75 C ATOM 246 C GLU A 62 −41.256 42.491 −1.122 1.00 89.37 C ATOM 247 O GLU A 62 −40.040 42.414 −1.313 1.00 90.84 O ATOM 248 CB GLU A 62 −42.699 43.219 −3.080 1.00 86.58 C ATOM 249 CG GLU A 62 −41.641 43.860 −3.967 1.00 87.94 C ATOM 250 CD GLU A 62 −42.234 44.940 −4.911 1.00 90.76 C ATOM 251 OE1 GLU A 62 −43.321 44.720 −5.505 1.00 96.67 O ATOM 252 OE2 GLU A 62 −41.611 46.016 −5.065 1.00 89.94 O ATOM 253 N ARG A 63 −41.798 42.964 0.003 1.00 90.60 N ATOM 254 CA ARG A 63 −41.036 43.318 1.207 1.00 88.93 C ATOM 255 C ARG A 63 −40.139 42.177 1.705 1.00 88.02 C ATOM 256 O ARG A 63 −39.007 42.409 2.131 1.00 88.49 O ATOM 257 CB ARG A 63 −42.028 43.691 2.309 1.00 91.49 C ATOM 258 CG ARG A 63 −41.443 43.980 3.698 1.00 95.03 C ATOM 259 CD ARG A 63 −42.573 44.340 4.643 1.00 99.84 C ATOM 260 NE ARG A 63 −42.113 44.841 5.939 1.00 103.70 N ATOM 261 CZ ARG A 63 −42.903 45.373 6.882 1.00 105.87 C ATOM 262 NH1 ARG A 63 −44.231 45.491 6.708 1.00 107.12 N ATOM 263 NH2 ARG A 63 −42.362 45.796 8.028 1.00 109.62 N ATOM 264 N LEU A 64 −40.658 40.955 1.648 1.00 85.74 N ATOM 265 CA LEU A 64 −39.932 39.775 2.097 1.00 82.11 C ATOM 266 C LEU A 64 −39.032 39.168 1.011 1.00 81.55 C ATOM 267 O LEU A 64 −38.378 38.184 1.272 1.00 78.38 O ATOM 268 CB LEU A 64 −40.923 38.712 2.602 1.00 80.82 C ATOM 269 CG LEU A 64 −41.851 39.090 3.769 1.00 79.80 C ATOM 270 CD1 LEU A 64 −42.899 37.999 4.007 1.00 70.96 C ATOM 271 CD2 LEU A 64 −41.056 39.337 5.038 1.00 73.56 C ATOM 272 N GLN A 65 −38.992 39.745 −0.198 1.00 82.04 N ATOM 273 CA GLN A 65 −38.174 39.189 −1.308 1.00 83.43 C ATOM 274 C GLN A 65 −36.720 39.672 −1.264 1.00 83.31 C ATOM 275 O GLN A 65 −36.305 40.518 −2.060 1.00 86.84 O ATOM 276 CB GLN A 65 −38.820 39.500 −2.662 1.00 82.54 C ATOM 277 CG GLN A 65 −40.120 38.749 −2.856 1.00 84.91 C ATOM 278 CD GLN A 65 −40.789 39.016 −4.182 1.00 88.24 C ATOM 279 OE1 GLN A 65 −40.516 40.018 −4.857 1.00 97.81 O ATOM 280 NE2 GLN A 65 −41.681 38.114 −4.569 1.00 97.75 N ATOM 281 N THR A 66 −35.973 39.113 −0.320 1.00 80.37 N ATOM 282 CA THR A 66 −34.561 39.413 −0.090 1.00 79.80 C ATOM 283 C THR A 66 −33.747 38.170 −0.355 1.00 79.26 C ATOM 284 O THR A 66 −34.306 37.105 −0.428 1.00 82.12 O ATOM 285 CB THR A 66 −34.334 39.778 1.353 1.00 79.66 C ATOM 286 OG1 THR A 66 −34.641 38.639 2.177 1.00 79.38 O ATOM 287 CG2 THR A 66 −35.218 40.953 1.753 1.00 75.58 C ATOM 288 N VAL A 67 −32.431 38.303 −0.492 1.00 76.86 N ATOM 289 CA VAL A 67 −31.561 37.138 −0.771 1.00 76.42 C ATOM 290 C VAL A 67 −31.679 36.080 0.342 1.00 76.58 C ATOM 291 O VAL A 67 −31.869 34.888 0.050 1.00 76.26 O ATOM 292 CB VAL A 67 −30.087 37.543 −0.956 1.00 77.31 C ATOM 293 CG1 VAL A 67 −29.185 36.325 −0.914 1.00 75.37 C ATOM 294 CG2 VAL A 67 −29.900 38.301 −2.270 1.00 72.45 C ATOM 295 N THR A 68 −31.568 36.512 1.597 1.00 71.98 N ATOM 296 CA THR A 68 −31.754 35.609 2.736 1.00 72.07 C ATOM 297 C THR A 68 −33.028 34.797 2.566 1.00 71.33 C ATOM 298 O THR A 68 −33.001 33.564 2.645 1.00 69.01 O ATOM 299 CB THR A 68 −31.810 36.372 4.062 1.00 71.58 C ATOM 300 OG1 THR A 68 −30.546 37.009 4.283 1.00 73.28 O ATOM 301 CG2 THR A 68 −32.098 35.420 5.213 1.00 71.59 C ATOM 302 N ASN A 69 −34.139 35.488 2.321 1.00 71.44 N ATOM 303 CA ASN A 69 −35.438 34.810 2.146 1.00 70.87 C ATOM 304 C ASN A 69 −35.534 33.842 0.957 1.00 71.14 C ATOM 305 O ASN A 69 −36.425 32.981 0.941 1.00 73.32 O ATOM 306 CB ASN A 69 −36.571 35.835 2.123 1.00 68.01 C ATOM 307 CG ASN A 69 −36.794 36.501 3.503 1.00 72.26 C ATOM 308 OD1 ASN A 69 −36.311 36.018 4.539 1.00 66.06 O ATOM 309 ND2 ASN A 69 −37.525 37.606 3.513 1.00 72.74 N ATOM 310 N TYR A 70 −34.640 33.969 −0.023 1.00 72.21 N ATOM 311 CA TYR A 70 −34.578 33.002 −1.133 1.00 73.01 C ATOM 312 C TYR A 70 −33.995 31.695 −0.599 1.00 73.34 C ATOM 313 O TYR A 70 −34.479 30.630 −0.942 1.00 77.66 O ATOM 314 CB TYR A 70 −33.781 33.529 −2.341 1.00 74.09 C ATOM 315 CG TYR A 70 −34.536 34.496 −3.245 1.00 75.84 C ATOM 316 CD1 TYR A 70 −35.265 35.553 −2.732 1.00 82.81 C ATOM 317 CD2 TYR A 70 −34.514 34.348 −4.620 1.00 84.86 C ATOM 318 CE1 TYR A 70 −35.957 36.444 −3.555 1.00 84.18 C ATOM 319 CE2 TYR A 70 −35.200 35.229 −5.453 1.00 85.94 C ATOM 320 CZ TYR A 70 −35.921 36.278 −4.918 1.00 84.44 C ATOM 321 OH TYR A 70 −36.603 37.149 −5.771 1.00 85.23 O ATOM 322 N PHE A 71 −32.961 31.788 0.238 1.00 75.67 N ATOM 323 CA PHE A 71 −32.384 30.609 0.904 1.00 73.91 C ATOM 324 C PHE A 71 −33.360 30.004 1.903 1.00 75.76 C ATOM 325 O PHE A 71 −33.426 28.779 2.046 1.00 72.03 O ATOM 326 CB PHE A 71 −31.108 30.949 1.666 1.00 74.63 C ATOM 327 CG PHE A 71 −30.024 31.535 0.817 1.00 72.11 C ATOM 328 CD1 PHE A 71 −29.584 30.876 −0.311 1.00 69.28 C ATOM 329 CD2 PHE A 71 −29.438 32.748 1.158 1.00 70.30 C ATOM 330 CE1 PHE A 71 −28.583 31.413 −1.095 1.00 74.96 C ATOM 331 CE2 PHE A 71 −28.449 33.285 0.396 1.00 72.37 C ATOM 332 CZ PHE A 71 −28.013 32.613 −0.749 1.00 76.19 C ATOM 333 N ILE A 72 −34.111 30.863 2.595 1.00 71.39 N ATOM 334 CA ILE A 72 −35.160 30.393 3.485 1.00 71.36 C ATOM 335 C ILE A 72 −36.192 29.597 2.692 1.00 73.02 C ATOM 336 O ILE A 72 −36.652 28.546 3.151 1.00 70.62 O ATOM 337 CB ILE A 72 −35.868 31.541 4.197 1.00 68.07 C ATOM 338 CG1 ILE A 72 −34.924 32.236 5.203 1.00 79.57 C ATOM 339 CG2 ILE A 72 −37.089 31.044 4.929 1.00 67.38 C ATOM 340 CD1 ILE A 72 −34.499 31.384 6.367 1.00 74.31 C ATOM 341 N THR A 73 −36.554 30.100 1.506 1.00 70.93 N ATOM 342 CA THR A 73 −37.500 29.397 0.644 1.00 71.88 C ATOM 343 C THR A 73 −36.937 28.040 0.264 1.00 73.34 C ATOM 344 O THR A 73 −37.663 27.045 0.296 1.00 77.13 O ATOM 345 CB THR A 73 −37.840 30.208 −0.618 1.00 73.11 C ATOM 346 OG1 THR A 73 −38.491 31.424 −0.228 1.00 71.91 O ATOM 347 CG2 THR A 73 −38.747 29.418 −1.548 1.00 72.28 C ATOM 348 N SER A 74 −35.654 28.000 −0.091 1.00 73.09 N ATOM 349 CA SER A 74 −34.981 26.740 −0.409 1.00 74.48 C ATOM 350 C SER A 74 −35.030 25.775 0.800 1.00 74.98 C ATOM 351 O SER A 74 −35.270 24.570 0.638 1.00 78.94 O ATOM 352 CB SER A 74 −33.546 27.014 −0.877 1.00 74.59 C ATOM 353 OG SER A 74 −32.820 25.829 −1.084 1.00 73.96 O ATOM 354 N LEU A 75 −34.810 26.318 1.995 1.00 73.74 N ATOM 355 CA LEU A 75 −34.911 25.567 3.255 1.00 72.08 C ATOM 356 C LEU A 75 −36.351 25.067 3.482 1.00 71.55 C ATOM 357 O LEU A 75 −36.565 23.897 3.819 1.00 71.52 O ATOM 358 CB LEU A 75 −34.437 26.455 4.418 1.00 72.86 C ATOM 359 CG LEU A 75 −34.085 25.884 5.771 1.00 78.35 C ATOM 360 CD1 LEU A 75 −33.026 24.797 5.661 1.00 80.50 C ATOM 361 CD2 LEU A 75 −33.614 27.061 6.695 1.00 77.36 C ATOM 362 N ALA A 76 −37.326 25.958 3.290 1.00 69.20 N ATOM 363 CA ALA A 76 −38.758 25.610 3.373 1.00 70.43 C ATOM 364 C ALA A 76 −39.206 24.520 2.362 1.00 70.45 C ATOM 365 O ALA A 76 −40.112 23.747 2.658 1.00 73.21 O ATOM 366 CB ALA A 76 −39.613 26.864 3.200 1.00 67.40 C ATOM 367 N CYS A 77 −38.578 24.461 1.184 1.00 72.62 N ATOM 368 CA CYS A 77 −38.911 23.411 0.176 1.00 72.54 C ATOM 369 C CYS A 77 −38.399 22.039 0.601 1.00 71.97 C ATOM 370 O CYS A 77 −39.084 21.047 0.401 1.00 75.84 O ATOM 371 CB CYS A 77 −38.350 23.752 −1.195 1.00 71.94 C ATOM 372 SG CYS A 77 −39.135 25.162 −1.970 1.00 78.85 S ATOM 373 N ALA A 78 −37.197 22.000 1.181 1.00 71.07 N ATOM 374 CA ALA A 78 −36.609 20.774 1.704 1.00 70.88 C ATOM 375 C ALA A 78 −37.457 20.206 2.846 1.00 71.74 C ATOM 376 O ALA A 78 −37.644 18.997 2.947 1.00 72.80 O ATOM 377 CB ALA A 78 −35.174 21.031 2.172 1.00 68.59 C ATOM 378 N ASP A 79 −37.964 21.083 3.698 1.00 71.88 N ATOM 379 CA ASP A 79 −38.859 20.676 4.783 1.00 71.72 C ATOM 380 C ASP A 79 −40.240 20.312 4.244 1.00 70.22 C ATOM 381 O ASP A 79 −40.872 19.384 4.743 1.00 70.23 O ATOM 382 CB ASP A 79 −38.934 21.775 5.847 1.00 68.87 C ATOM 383 CG ASP A 79 −37.609 21.931 6.630 1.00 82.57 C ATOM 384 OD1 ASP A 79 −36.914 20.899 6.889 1.00 83.92 O ATOM 385 OD2 ASP A 79 −37.258 23.081 6.992 1.00 79.92 O ATOM 386 N LEU A 80 −40.712 21.034 3.237 1.00 67.28 N ATOM 387 CA LEU A 80 −42.008 20.683 2.616 1.00 68.96 C ATOM 388 C LEU A 80 −41.960 19.284 2.009 1.00 65.98 C ATOM 389 O LEU A 80 −42.889 18.505 2.184 1.00 70.25 O ATOM 390 CB LEU A 80 −42.430 21.705 1.562 1.00 63.48 C ATOM 391 CG LEU A 80 −43.828 21.494 0.969 1.00 70.22 C ATOM 392 CD1 LEU A 80 −44.896 21.346 2.048 1.00 68.20 C ATOM 393 CD2 LEU A 80 −44.193 22.645 −0.015 1.00 74.86 C ATOM 394 N VAL A 81 −40.877 18.972 1.302 1.00 66.65 N ATOM 395 CA VAL A 81 −40.686 17.628 0.713 1.00 68.44 C ATOM 396 C VAL A 81 −40.559 16.537 1.815 1.00 68.93 C ATOM 397 O VAL A 81 −41.066 15.426 1.647 1.00 71.60 O ATOM 398 CB VAL A 81 −39.475 17.608 −0.253 1.00 68.36 C ATOM 399 CG1 VAL A 81 −39.202 16.216 −0.740 1.00 65.96 C ATOM 400 CG2 VAL A 81 −39.713 18.581 −1.445 1.00 63.74 C ATOM 401 N MET A 82 −39.889 16.859 2.923 1.00 67.67 N ATOM 402 CA MET A 82 −39.862 15.968 4.099 1.00 69.67 C ATOM 403 C MET A 82 −41.282 15.658 4.578 1.00 67.76 C ATOM 404 O MET A 82 −41.632 14.503 4.804 1.00 66.68 O ATOM 405 CB MET A 82 −39.088 16.603 5.245 1.00 70.60 C ATOM 406 CG MET A 82 −37.580 16.550 5.089 1.00 76.37 C ATOM 407 SD MET A 82 −36.812 15.024 5.666 1.00 86.09 S ATOM 408 CE MET A 82 −37.447 15.024 7.316 1.00 88.14 C ATOM 409 N GLY A 83 −42.089 16.703 4.725 1.00 66.27 N ATOM 410 CA GLY A 83 −43.480 16.564 5.170 1.00 68.36 C ATOM 411 C GLY A 83 −44.416 15.795 4.250 1.00 71.02 C ATOM 412 O GLY A 83 −45.350 15.161 4.730 1.00 72.14 O ATOM 413 N LEU A 84 −44.169 15.853 2.939 1.00 71.19 N ATOM 414 CA LEU A 84 −45.010 15.170 1.939 1.00 73.59 C ATOM 415 C LEU A 84 −44.469 13.816 1.460 1.00 73.92 C ATOM 416 O LEU A 84 −45.224 12.859 1.294 1.00 72.70 O ATOM 417 CB LEU A 84 −45.195 16.069 0.702 1.00 72.52 C ATOM 418 CG LEU A 84 −45.901 17.406 0.885 1.00 75.09 C ATOM 419 CD1 LEU A 84 −45.933 18.147 −0.445 1.00 78.12 C ATOM 420 CD2 LEU A 84 −47.316 17.232 1.430 1.00 77.53 C ATOM 421 N ALA A 85 −43.166 13.751 1.237 1.00 73.21 N ATOM 422 CA ALA A 85 −42.540 12.584 0.628 1.00 73.60 C ATOM 423 C ALA A 85 −41.858 11.673 1.615 1.00 72.66 C ATOM 424 O ALA A 85 −41.825 10.482 1.377 1.00 78.23 O ATOM 425 CB ALA A 85 −41.533 13.043 −0.426 1.00 74.36 C ATOM 426 N VAL A 86 −41.319 12.218 2.710 1.00 74.33 N ATOM 427 CA VAL A 86 −40.544 11.419 3.678 1.00 71.85 C ATOM 428 C VAL A 86 −41.349 11.025 4.910 1.00 71.02 C ATOM 429 O VAL A 86 −41.584 9.860 5.132 1.00 73.11 O ATOM 430 CB VAL A 86 −39.254 12.136 4.110 1.00 71.51 C ATOM 431 CG1 VAL A 86 −38.400 11.213 4.969 1.00 68.65 C ATOM 432 CG2 VAL A 86 −38.467 12.587 2.885 1.00 70.81 C ATOM 433 N VAL A 87 −41.773 11.995 5.707 1.00 71.04 N ATOM 434 CA VAL A 87 −42.455 11.692 7.002 1.00 71.84 C ATOM 435 C VAL A 87 −43.718 10.799 6.915 1.00 72.51 C ATOM 436 O VAL A 87 −43.894 9.940 7.783 1.00 76.50 O ATOM 437 CB VAL A 87 −42.798 12.999 7.819 1.00 72.70 C ATOM 438 CG1 VAL A 87 −43.506 12.655 9.148 1.00 65.72 C ATOM 439 CG2 VAL A 87 −41.549 13.826 8.093 1.00 69.80 C ATOM 440 N PRO A 88 −44.588 10.984 5.886 1.00 74.57 N ATOM 441 CA PRO A 88 −45.792 10.131 5.828 1.00 73.89 C ATOM 442 C PRO A 88 −45.514 8.629 5.696 1.00 74.31 C ATOM 443 O PRO A 88 −46.204 7.831 6.322 1.00 72.95 O ATOM 444 CB PRO A 88 −46.529 10.654 4.580 1.00 72.19 C ATOM 445 CG PRO A 88 −46.040 12.026 4.413 1.00 70.91 C ATOM 446 CD PRO A 88 −44.581 11.922 4.748 1.00 75.19 C ATOM 447 N PHE A 89 −41.517 8.266 4.895 1.00 75.27 N ATOM 448 CA PHE A 89 −44.153 6.857 4.707 1.00 76.82 C ATOM 449 C PHE A 89 −43.325 6.300 5.850 1.00 75.58 C ATOM 450 O PHE A 89 −43.466 5.124 6.182 1.00 76.46 O ATOM 451 CB PHE A 89 −43.434 6.665 3.380 1.00 81.35 C ATOM 452 CG PHE A 89 −44.258 7.080 2.177 1.00 87.88 C ATOM 453 CD1 PHE A 89 −45.600 6.699 2.046 1.00 93.20 C ATOM 454 CD2 PHE A 89 −43.709 7.840 1.186 1.00 87.00 C ATOM 455 CE1 PHE A 89 −46.350 7.082 0.940 1.00 93.35 C ATOM 456 CE2 PHE A 89 −44.469 8.219 0.076 1.00 92.27 C ATOM 457 CZ PHE A 89 −45.780 7.839 −0.040 1.00 91.97 C ATOM 458 N GLY A 90 −42.468 7.125 6.444 1.00 74.71 N ATOM 459 CA GLY A 90 −41.738 6.731 7.650 1.00 75.06 C ATOM 460 C GLY A 90 −42.728 6.369 8.749 1.00 74.16 C ATOM 461 O GLY A 90 −42.572 5.341 9.402 1.00 77.84 O ATOM 462 N ALA A 91 −43.747 7.223 8.939 1.00 72.72 N ATOM 463 CA ALA A 91 −44.835 6.994 9.912 1.00 72.43 C ATOM 464 C ALA A 91 −45.543 5.664 9.680 1.00 74.22 C ATOM 465 O ALA A 91 −45.668 4.853 10.604 1.00 74.73 O ATOM 466 CB ALA A 91 −45.849 8.129 9.849 1.00 69.78 C ATOM 467 N ALA A 92 −45.999 5.464 8.437 1.00 73.35 N ATOM 468 CA ALA A 92 −46.699 4.241 8.013 1.00 74.78 C ATOM 469 C ALA A 92 −45.850 2.998 8.219 1.00 73.86 C ATOM 470 O ALA A 92 −46.355 1.985 8.671 1.00 76.34 O ATOM 471 CB ALA A 92 −47.131 4.348 6.541 1.00 73.00 C ATOM 472 N HIS A 93 −44.566 3.098 7.881 1.00 75.22 N ATOM 473 CA HIS A 93 −43.592 2.022 8.080 1.00 75.74 C ATOM 474 C HIS A 93 −43.521 1.575 9.543 1.00 76.69 C ATOM 475 O HIS A 93 −43.429 0.383 9.818 1.00 80.31 O ATOM 476 CB HIS A 93 −42.216 2.487 7.579 1.00 77.29 C ATOM 477 CG HIS A 93 −41.151 1.442 7.633 1.00 75.35 C ATOM 478 ND1 HIS A 93 −39.816 1.752 7.759 1.00 74.50 N ATOM 479 CD2 HIS A 93 −41.220 0.094 7.579 1.00 76.44 C ATOM 480 CE1 HIS A 93 −39.110 0.635 7.779 1.00 78.85 C ATOM 481 NE2 HIS A 93 −39.939 −0.386 7.671 1.00 74.19 N ATOM 482 N ILE A 94 −43.568 2.533 10.464 1.00 77.24 N ATOM 483 CA ILE A 94 −43.534 2.243 11.901 1.00 77.99 C ATOM 484 C ILE A 94 −44.855 1.668 12.417 1.00 78.57 C ATOM 485 O ILE A 94 −44.853 0.680 13.162 1.00 80.24 O ATOM 486 CB ILE A 94 −43.193 3.506 12.720 1.00 78.41 C ATOM 487 CG1 ILE A 94 −41.745 3.911 12.482 1.00 78.28 C ATOM 488 CG2 ILE A 94 −43.404 3.262 14.201 1.00 79.28 C ATOM 489 CD1 ILE A 94 −41.379 5.210 13.108 1.00 77.39 C ATOM 490 N LEU A 95 −45.967 2.283 12.024 1.00 78.45 N ATOM 491 CA LEU A 95 −47.302 1.855 12.478 1.00 79.09 C ATOM 492 C LEU A 95 −47.729 0.492 11.925 1.00 80.71 C ATOM 493 O LEU A 95 −48.401 −0.271 12.626 1.00 82.74 O ATOM 494 CB LEU A 95 −48.347 2.916 12.119 1.00 79.29 C ATOM 495 CG LEU A 95 −48.202 4.255 12.843 1.00 78.03 C ATOM 496 CD1 LEU A 95 −49.033 5.334 12.164 1.00 77.81 C ATOM 497 CD2 LEU A 95 −48.587 4.110 14.306 1.00 75.72 C ATOM 498 N MET A 96 −47.346 0.195 10.685 1.00 80.6 N ATOM 499 CA MET A 96 −47.635 −1.103 10.066 1.00 80.57 C ATOM 500 C MET A 96 −46.592 −2.157 10.421 1.00 78.70 C ATOM 501 O MET A 96 −46.849 −3.336 10.242 1.00 75.20 O ATOM 502 CB MET A 96 −47.727 −0.965 8.552 1.00 80.83 C ATOM 503 CG MET A 96 −48.948 −0.212 8.114 1.00 83.20 C ATOM 504 SD MET A 96 −48.947 0.080 6.357 1.00 91.34 S ATOM 505 CE MET A 96 −50.698 0.372 6.060 1.00 92.81 C ATOM 506 N LYS A 97 −45.429 −1.717 10.918 1.00 80.00 N ATOM 507 CA LYS A 97 −44.302 −2.598 11.317 1.00 81.41 C ATOM 508 C LYS A 97 −43.720 −3.385 10.128 1.00 79.08 C ATOM 509 O LYS A 97 −43.216 −4.494 10.294 1.00 78.90 O ATOM 510 CB LYS A 97 −44.705 −3.535 12.483 1.00 82.51 C ATOM 511 CG LYS A 97 −45.084 −2.800 13.750 1.00 85.16 C ATOM 512 CD LYS A 97 −45.946 −3.663 14.664 1.00 86.38 C ATOM 513 CE LYS A 97 −46.262 −2.937 15.971 1.00 90.74 C ATOM 514 NZ LYS A 97 −46.864 −1.569 15.763 1.00 92.55 N ATOM 515 N MET A 98 −43.797 −2.788 8.941 1.00 78.37 N ATOM 516 CA MET A 98 −43.317 −3.387 7.703 1.00 78.62 C ATOM 517 C MET A 98 −43.508 −2.378 6.592 1.00 78.05 C ATOM 518 O MET A 98 −44.326 −1.458 6.720 1.00 80.11 O ATOM 519 CB MET A 98 −44.045 −4.706 7.375 1.00 79.92 C ATOM 520 CG MET A 98 −45.573 −4.628 7.326 1.00 84.42 C ATOM 521 SD MET A 98 −46.226 −4.350 5.681 1.00 96.30 S ATOM 522 CE MET A 98 −45.936 −5.962 4.938 1.00 95.48 C ATOM 523 N TRP A 99 −42.754 −2.552 5.510 1.00 77.49 N ATOM 524 CA TRP A 99 −42.823 −1.681 4.333 1.00 75.69 C ATOM 525 C TRP A 99 −43.856 −2.268 3.372 1.00 73.64 C ATOM 526 O TRP A 99 −43.802 −3.441 3.079 1.00 77.31 O ATOM 527 CB TRP A 99 −41.444 −1.587 3.691 1.00 74.33 C ATOM 528 CG TRP A 99 −41.339 −0.599 2.633 1.00 74.05 C ATOM 529 CD1 TRP A 99 −41.293 −0.838 1.306 1.00 70.83 C ATOM 530 CD2 TRP A 99 −41.262 0.821 2.788 1.00 73.53 C ATOM 531 NE1 TRP A 99 −41.195 0.331 0.624 1.00 73.91 N ATOM 532 CE2 TRP A 99 −41.172 1.368 1.505 1.00 73.77 C ATOM 533 CE3 TRP A 99 −41.262 1.680 3.887 1.00 74.17 C ATOM 534 CZ2 TRP A 99 −41.083 2.737 1.277 1.00 75.99 C ATOM 535 CZ3 TRP A 99 −41.172 3.054 3.664 1.00 71.44 C ATOM 536 CH2 TRP A 99 −41.083 3.564 2.370 1.00 77.11 C ATOM 537 N THR A 100 −44.795 −1.451 2.892 1.00 73.03 N ATOM 538 CA THR A 100 −45.840 −1.896 1.924 1.00 72.98 C ATOM 539 C THR A 100 −45.766 −1.236 0.546 1.00 71.23 C ATOM 540 O THR A 100 −46.562 −1.571 −0.333 1.00 71.17 O ATOM 541 CB THR A 100 −47.266 −1.597 2.444 1.00 73.76 C ATOM 542 OG1 THR A 100 −47.509 −0.178 2.405 1.00 76.78 O ATOM 543 CG2 THR A 100 −47.446 −2.076 3.842 1.00 78.02 C ATOM 544 N PHE A 101 −44.829 −0.312 0.355 1.00 70.30 N ATOM 545 CA PHE A 101 −44.748 0.499 −0.870 1.00 72.26 C ATOM 546 C PHE A 101 −43.772 −0.083 −1.920 1.00 74.09 C ATOM 547 O PHE A 101 −43.621 0.485 −3.018 1.00 74.48 O ATOM 548 CB PHE A 101 −44.414 1.947 −0.481 1.00 72.34 C ATOM 549 CG PHE A 101 −45.268 2.457 0.663 1.00 70.71 C ATOM 550 CD1 PHE A 101 −44.766 2.495 1.965 1.00 71.75 C ATOM 551 CD2 PHE A 101 −46.572 2.887 0.438 1.00 68.75 C ATOM 552 CE1 PHE A 101 −45.552 2.961 3.028 1.00 71.54 C ATOM 553 CE2 PHE A 101 −47.364 3.354 1.493 1.00 70.01 C ATOM 554 CZ PHE A 101 −46.853 3.391 2.789 1.00 68.99 C ATOM 555 N GLY A 102 −43.122 −1.207 −1.581 1.00 73.14 N ATOM 556 CA GLY A 102 −42.234 −1.922 −2.502 1.00 73.21 C ATOM 557 C GLY A 102 −40.821 −1.391 −2.546 1.00 72.86 C ATOM 558 O GLY A 102 −40.552 −0.299 −2.091 1.00 72.40 O ATOM 559 N ASN A 103 −39.916 −2.184 −3.114 1.00 74.31 N ATOM 560 CA ASN A 103 −38.484 −1.824 −3.165 1.00 74.88 C ATOM 561 C ASN A 103 −38.129 −0.574 −3.956 1.00 74.74 C ATOM 562 O ASN A 103 −37.251 0.175 −3.515 1.00 76.17 O ATOM 563 CB ASN A 103 −37.626 −2.998 −3.690 1.00 74.34 C ATOM 564 CG ASN A 103 −37.420 −4.108 −2.651 1.00 74.79 C ATOM 565 OD1 ASN A 103 −37.242 −5.271 −3.012 1.00 76.47 O ATOM 566 ND2 ASN A 103 −37.444 −3.753 −1.363 1.00 74.57 N ATOM 567 N PHE A 104 −38.782 −0.339 −5.098 1.00 74.65 N ATOM 568 CA PHE A 104 −38.478 0.860 −5.884 1.00 75.28 C ATOM 569 C PHE A 104 −38.644 2.100 −5.041 1.00 75.71 C ATOM 570 O PHE A 104 −37.710 2.895 −4.906 1.00 77.93 O ATOM 571 CB PHE A 104 −39.367 1.020 −7.116 1.00 78.02 C ATOM 572 CG PHE A 104 −39.279 2.408 −7.727 1.00 79.37 C ATOM 573 CD1 PHE A 104 −38.129 2.802 −8.421 1.00 80.42 C ATOM 574 CD2 PHE A 104 −40.337 3.321 −7.603 1.00 81.51 C ATOM 575 CE1 PHE A 104 −38.031 4.072 −8.984 1.00 79.83 C ATOM 576 CE2 PHE A 104 −40.248 4.598 −8.164 1.00 83.42 C ATOM 577 CZ PHE A 104 −39.091 4.975 −8.857 1.00 82.52 C ATOM 578 N TRP A 105 −39.839 2.255 −4.478 1.00 76.21 N ATOM 579 CA TRP A 105 −40.134 3.416 −3.670 1.00 75.91 C ATOM 580 C TRP A 105 −39.295 3.456 −2.357 1.00 76.37 C ATOM 581 O TRP A 105 −38.972 4.534 −1.899 1.00 74.11 O ATOM 582 CB TRP A 105 −41.645 3.562 −3.386 1.00 78.05 C ATOM 583 CG TRP A 105 −41.870 4.799 −2.623 1.00 82.40 C ATOM 584 CD1 TRP A 105 −42.157 4.889 −1.318 1.00 83.43 C ATOM 585 CD2 TRP A 105 −41.810 6.147 −3.120 1.00 87.60 C ATOM 586 NE1 TRP A 105 −42.288 6.182 −0.950 1.00 83.52 N ATOM 587 CE2 TRP A 105 −42.082 6.994 −2.033 1.00 84.67 C ATOM 588 CE3 TRP A 105 −41.556 6.716 −4.381 1.00 86.42 C ATOM 589 CZ2 TRP A 105 −42.109 8.401 −2.155 1.00 81.05 C ATOM 590 CZ3 TRP A 105 −41.583 8.115 −4.507 1.00 84.49 C ATOM 591 CH2 TRP A 105 −41.860 8.938 −3.391 1.00 85.45 C ATOM 592 N CYS A 106 −38.952 2.302 −1.775 1.00 74.15 N ATOM 593 CA CYS A 106 −38.086 2.278 −0.573 1.00 75.23 C ATOM 594 C CYS A 106 −36.763 2.982 −0.834 1.00 74.63 C ATOM 595 O CYS A 106 −36.332 3.831 −0.045 1.00 72.22 O ATOM 596 CB CYS A 106 −37.827 0.848 −0.097 1.00 75.82 C ATOM 597 SG CYS A 106 −36.530 0.694 1.167 1.00 75.27 S ATOM 598 N GLU A 107 −36.137 2.618 −1.946 1.00 74.18 N ATOM 599 CA GLU A 107 −34.882 3.219 −2.380 1.00 73.95 C ATOM 600 C GLU A 107 −35.054 4.698 −2.773 1.00 73.77 C ATOM 601 O GLU A 107 −34.185 5.521 −2.469 1.00 69.11 O ATOM 602 CB GLU A 107 −34.337 2.411 −3.542 1.00 74.75 C ATOM 603 CG GLU A 107 −33.001 2.841 −4.117 1.00 78.43 C ATOM 604 CD GLU A 107 −32.677 2.072 −5.397 1.00 82.58 C ATOM 605 OE1 GLU A 107 −32.915 0.843 −5.452 1.00 87.52 O ATOM 606 OE2 GLU A 107 −32.186 2.688 −6.352 1.00 90.13 O ATOM 607 N PHE A 108 −36.166 5.025 −3.445 1.00 72.86 N ATOM 608 CA PHE A 108 −36.458 6.414 −3.816 1.00 70.73 C ATOM 609 C PHE A 108 −36.705 7.236 −2.556 1.00 68.58 C ATOM 610 O PHE A 108 −36.073 8.275 −2.357 1.00 66.92 O ATOM 611 CB PHE A 108 −37.676 6.506 −4.729 1.00 70.86 C ATOM 612 CG PHE A 108 −37.744 7.783 −5.526 1.00 72.80 C ATOM 613 CD1 PHE A 108 −37.339 7.799 −6.868 1.00 78.82 C ATOM 614 CD2 PHE A 108 −38.205 8.965 −4.961 1.00 74.96 C ATOM 615 CE1 PHE A 108 −37.396 8.977 −7.621 1.00 76.80 C ATOM 616 CE2 PHE A 108 −38.262 10.149 −5.720 1.00 74.68 C ATOM 617 CZ PHE A 108 −37.858 10.148 −7.043 1.00 76.41 C ATOM 618 N TRP A 109 −37.628 6.752 −1.723 1.00 66.71 N ATOM 619 CA TRP A 109 −37.960 7.358 −0.431 1.00 68.89 C ATOM 620 C TRP A 109 −36.701 7.635 0.361 1.00 69.04 C ATOM 621 O TRP A 109 −36.504 8.752 0.807 1.00 68.71 O ATOM 622 CB TRP A 109 −38.866 6.437 0.371 1.00 71.86 C ATOM 623 CG TRP A 109 −39.102 6.855 1.747 1.00 70.49 C ATOM 624 CD1 TRP A 109 −39.862 7.878 2.149 1.00 72.33 C ATOM 625 CD2 TRP A 109 −38.569 6.248 2.938 1.00 74.86 C ATOM 626 NE1 TRP A 109 −39.847 7.967 3.516 1.00 76.09 N ATOM 627 CE2 TRP A 109 −39.062 6.979 4.025 1.00 72.44 C ATOM 628 CE3 TRP A 109 −37.720 5.156 3.187 1.00 77.42 C ATOM 629 CZ2 TRP A 109 −38.747 6.669 5.347 1.00 75.85 C ATOM 630 CZ3 TRP A 109 −37.404 4.849 4.532 1.00 74.21 C ATOM 631 CH2 TRP A 109 −37.919 5.602 5.575 1.00 73.66 C ATOM 632 N THR A 110 −35.861 6.607 0.518 1.00 64.06 N ATOM 633 CA THR A 110 −34.570 6.747 1.190 1.00 66.65 C ATOM 634 C THR A 110 −33.707 7.863 0.621 1.00 68.41 C ATOM 635 O THR A 110 −33.116 8.624 1.385 1.00 71.70 O ATOM 636 CB THR A 110 −33.757 5.477 1.102 1.00 66.89 C ATOM 637 OG1 THR A 110 −34.491 4.404 1.699 1.00 70.60 O ATOM 638 CG2 THR A 110 −32.427 5.645 1.830 1.00 64.09 C ATOM 639 N SER A 111 −33.636 7.959 −0.709 1.00 69.98 N ATOM 640 CA SER A 111 −32.849 9.017 −1.387 1.00 70.65 C ATOM 641 C SER A 111 −33.343 10.443 −1.092 1.00 73.21 C ATOM 642 O SER A 111 −32.524 11.356 −0.899 1.00 76.91 O ATOM 643 CB SER A 111 −32.825 8.802 −2.909 1.00 72.35 C ATOM 644 OG SER A 111 −32.247 7.565 −3.262 1.00 69.46 O ATOM 645 N ILE A 112 −34.669 10.626 −1.063 1.00 72.31 N ATOM 646 CA ILE A 112 −35.290 11.938 −0.786 1.00 70.06 C ATOM 647 C ILE A 112 −35.011 12.365 0.650 1.00 69.36 C ATOM 648 O ILE A 112 −34.706 13.535 0.916 1.00 68.07 O ATOM 649 CB ILE A 112 −36.819 11.916 −1.006 1.00 68.74 C ATOM 650 CG1 ILE A 112 −37.174 11.632 −2.476 1.00 75.90 C ATOM 651 CG2 ILE A 112 −37.426 13.234 −0.645 1.00 72.66 C ATOM 652 CD1 ILE A 112 −38.665 11.518 −2.704 1.00 73.21 C ATOM 653 N ASP A 113 −35.126 11.403 1.557 1.00 68.09 N ATOM 654 CA ASP A 113 −34.823 11.584 2.973 1.00 69.93 C ATOM 655 C ASP A 113 −33.435 12.218 3.102 1.00 68.24 C ATOM 656 O ASP A 113 −33.311 13.328 3.600 1.00 69.08 O ATOM 657 CB ASP A 113 −34.916 10.225 3.681 1.00 71.22 C ATOM 658 CG ASP A 113 −35.001 10.325 5.163 1.00 71.94 C ATOM 659 OD1 ASP A 113 −34.453 11.263 5.771 1.00 73.99 O ATOM 660 OD2 ASP A 113 −35.644 9.419 5.727 1.00 78.39 O ATOM 661 N VAL A 114 −32.415 11.501 2.640 1.00 69.76 N ATOM 662 CA VAL A 114 −31.016 11.951 2.687 1.00 69.79 C ATOM 663 C VAL A 114 −30.790 13.289 1.934 1.00 70.38 C ATOM 664 O VAL A 114 −30.022 14.132 2.409 1.00 66.61 O ATOM 665 CB VAL A 114 −30.070 10.827 2.158 1.00 71.48 C ATOM 666 CG1 VAL A 114 −28.657 11.338 1.891 1.00 71.98 C ATOM 667 CG2 VAL A 114 −30.054 9.643 3.147 1.00 68.67 C ATOM 668 N LEU A 115 −31.455 13.467 0.780 1.00 69.66 N ATOM 669 CA LEU A 115 −31.356 14.717 −0.015 1.00 70.60 C ATOM 670 C LEU A 115 −31.817 15.929 0.773 1.00 72.08 C ATOM 671 O LEU A 115 −31.084 16.909 0.895 1.00 71.75 O ATOM 672 CB LEU A 115 −32.193 14.621 −1.314 1.00 73.84 C ATOM 673 CG LEU A 115 −32.357 15.859 −2.238 1.00 73.55 C ATOM 674 CD1 LEU A 115 −31.014 16.399 −2.735 1.00 72.28 C ATOM 675 CD2 LEU A 115 −33.278 15.542 −3.431 1.00 66.35 C ATOM 676 N CYS A 116 −33.039 15.841 1.297 1.00 72.44 N ATOM 677 CA CYS A 116 −33.670 16.941 2.034 1.00 73.77 C ATOM 678 C CYS A 116 −32.868 17.364 3.251 1.00 74.35 C ATOM 679 O CYS A 116 −32.711 18.570 3.492 1.00 75.14 O ATOM 680 CB CYS A 116 −35.112 16.586 2.437 1.00 72.52 C ATOM 681 SG CYS A 116 −36.268 16.557 1.013 1.00 80.97 S ATOM 682 N VAL A 117 −32.365 16.391 4.010 1.00 72.36 N ATOM 683 CA VAL A 117 −31.509 16.695 5.167 1.00 73.25 C ATOM 684 C VAL A 117 −30.188 17.351 4.725 1.00 75.09 C ATOM 685 O VAL A 117 −29.729 18.311 5.369 1.00 76.15 O ATOM 686 CB VAL A 117 −31.217 15.439 6.020 1.00 73.55 C ATOM 687 CG1 VAL A 117 −30.199 15.737 7.098 1.00 69.89 C ATOM 688 CG2 VAL A 117 −32.503 14.916 6.641 1.00 73.99 C ATOM 689 N THR A 118 −29.592 16.837 3.635 1.00 71.84 N ATOM 690 CA THR A 118 −28.329 17.366 3.096 1.00 71.32 C ATOM 691 C THR A 118 −28.501 18.788 2.575 1.00 73.72 C ATOM 692 O THR A 118 −27.710 19.656 2.916 1.00 78.08 O ATOM 693 CB THR A 118 −27.767 16.482 1.949 1.00 72.20 C ATOM 694 OG1 THR A 118 −27.512 15.161 2.433 1.00 75.03 O ATOM 695 CG2 THR A 118 −26.471 17.066 1.389 1.00 65.55 C ATOM 696 N ALA A 119 −29.534 19.013 1.752 1.00 72.84 N ATOM 697 CA ALA A 119 −29.857 20.351 1.220 1.00 71.95 C ATOM 698 C ALA A 119 −30.142 21.361 2.334 1.00 71.48 C ATOM 699 O ALA A 119 −29.750 22.521 2.226 1.00 73.66 O ATOM 700 CB ALA A 119 −31.054 20.284 0.243 1.00 69.50 C ATOM 701 N SER A 120 −30.818 20.920 3.394 1.00 71.79 N ATOM 702 CA SER A 120 −31.124 21.803 4.531 1.00 71.94 C ATOM 703 C SER A 120 −29.861 22.374 5.153 1.00 73.64 C ATOM 704 O SER A 120 −29.724 23.598 5.252 1.00 74.21 O ATOM 705 CB SER A 120 −31.927 21.071 5.584 1.00 71.95 C ATOM 706 OG SER A 120 −33.202 20.758 5.077 1.00 75.41 O ATOM 707 N ILE A 121 −28.945 21.500 5.562 1.00 74.66 N ATOM 708 CA ILE A 121 −27.694 21.946 6.223 1.00 72.09 C ATOM 709 C ILE A 121 −26.767 22.684 5.253 1.00 74.47 C ATOM 710 O ILE A 121 −26.066 23.606 5.668 1.00 72.06 O ATOM 711 CB ILE A 121 −26.922 20.782 6.903 1.00 72.33 C ATOM 712 CG1 ILE A 121 −25.776 21.297 7.823 1.00 75.07 C ATOM 713 CG2 ILE A 121 −26.349 19.826 5.858 1.00 69.34 C ATOM 714 CD1 ILE A 121 −26.149 22.421 8.816 1.00 75.41 C ATOM 715 N GLU A 122 −26.758 22.289 3.977 1.00 71.61 N ATOM 716 CA GLU A 122 −25.957 23.013 2.976 1.00 73.61 C ATOM 717 C GLU A 122 −26.477 24.430 2.815 1.00 71.97 C ATOM 718 O GLU A 122 −25.684 25.357 2.719 1.00 73.76 O ATOM 719 CB GLU A 122 −25.936 22.298 1.619 1.00 71.23 C ATOM 720 CG GLU A 122 −25.042 21.050 1.616 1.00 78.82 C ATOM 721 CD GLU A 122 −24.898 20.393 0.249 1.00 76.68 C ATOM 722 OE1 GLU A 122 −25.210 21.034 −0.777 1.00 81.34 O ATOM 723 OE2 GLU A 122 −24.464 19.221 0.208 1.00 82.37 O ATOM 724 N THR A 123 −27.804 24.585 2.788 1.00 71.94 N ATOM 725 CA THR A 123 −28.437 25.916 2.693 1.00 71.90 C ATOM 726 C THR A 123 −28.141 26.762 3.909 1.00 71.32 C ATOM 727 O THR A 123 −27.722 27.903 3.768 1.00 70.52 O ATOM 728 CB THR A 123 −29.940 25.819 2.596 1.00 69.91 C ATOM 729 OG1 THR A 123 −30.275 24.924 1.534 1.00 70.66 O ATOM 730 CG2 THR A 123 −30.539 27.214 2.354 1.00 68.19 C ATOM 731 N LEU A 124 −28.364 26.197 5.099 1.00 69.53 N ATOM 732 CA LEU A 124 −28.044 26.877 6.330 1.00 70.49 C ATOM 733 C LEU A 124 −26.624 27.353 6.294 1.00 69.98 C ATOM 734 O LEU A 124 −26.349 28.465 6.684 1.00 72.08 O ATOM 735 CB LEU A 124 −28.230 25.963 7.561 1.00 72.66 C ATOM 736 CG LEU A 124 −29.654 25.655 7.991 1.00 71.44 C ATOM 737 CD1 LEU A 124 −29.643 24.635 9.128 1.00 74.54 C ATOM 738 CD2 LEU A 124 −30.358 26.930 8.384 1.00 68.04 C ATOM 739 N CYS A 125 −25.726 26.509 5.822 1.00 72.50 N ATOM 740 CA CYS A 125 −24.319 26.880 5.727 1.00 75.56 C ATOM 741 C CYS A 125 −24.087 28.030 4.746 1.00 73.80 C ATOM 742 O CYS A 125 −23.222 28.868 4.985 1.00 74.28 O ATOM 743 CB CYS A 125 −23.474 25.682 5.322 1.00 76.67 C ATOM 744 SG CYS A 125 −21.781 25.957 5.620 1.00 84.21 S ATOM 745 N VAL A 126 −24.860 28.059 3.652 1.00 76.58 N ATOM 746 CA VAL A 126 −24.803 29.161 2.654 1.00 74.32 C ATOM 747 C VAL A 126 −25.327 30.458 3.269 1.00 74.06 C ATOM 748 O VAL A 126 −24.820 31.529 2.959 1.00 74.99 O ATOM 749 CB VAL A 126 −25.612 28.848 1.381 1.00 73.89 C ATOM 750 CG1 VAL A 126 −25.614 30.050 0.435 1.00 78.42 C ATOM 751 CG2 VAL A 126 −25.056 27.612 0.682 1.00 75.52 C ATOM 752 N ILE A 127 −26.341 30.352 4.138 1.00 73.19 N ATOM 753 CA ILE A 127 −26.902 31.527 4.840 1.00 71.30 C ATOM 754 C ILE A 127 −25.846 32.137 5.724 1.00 69.71 C ATOM 755 O ILE A 127 −25.706 33.342 5.750 1.00 73.08 O ATOM 756 CB ILE A 127 −28.150 31.200 5.671 1.00 70.82 C ATOM 757 CG1 ILE A 127 −29.301 30.720 4.782 1.00 74.13 C ATOM 758 CG2 ILE A 127 −28.636 32.429 6.410 1.00 72.22 C ATOM 759 CD1 ILE A 127 −30.450 30.161 5.540 1.00 73.97 C ATOM 760 N ALA A 128 −25.105 31.298 6.440 1.00 70.03 N ATOM 761 CA ALA A 128 −24.031 31.756 7.323 1.00 70.78 C ATOM 762 C ALA A 128 −22.929 32.481 6.577 1.00 70.23 C ATOM 763 O ALA A 128 −22.493 33.544 7.004 1.00 70.16 O ATOM 764 CB ALA A 128 −23.443 30.585 8.092 1.00 72.00 C ATOM 765 N VAL A 129 −22.480 31.905 5.463 1.00 73.67 N ATOM 766 CA VAL A 129 −21.415 32.529 4.657 1.00 70.68 C ATOM 767 C VAL A 129 −21.923 33.853 4.062 1.00 70.47 C ATOM 768 O VAL A 129 −21.212 34.853 4.092 1.00 73.60 O ATOM 769 CB VAL A 129 −20.878 31.595 3.556 1.00 70.74 C ATOM 770 CG1 VAL A 129 −19.835 32.328 2.689 1.00 65.41 C ATOM 771 CG2 VAL A 129 −20.285 30.331 4.172 1.00 66.69 C ATOM 772 N ASP A 130 −23.149 33.836 3.536 1.00 72.22 N ATOM 773 CA ASP A 130 −23.812 35.034 2.993 1.00 69.02 C ATOM 774 C ASP A 130 −23.965 36.125 4.036 1.00 68.79 C ATOM 775 O ASP A 130 −23.668 37.270 3.758 1.00 66.69 O ATOM 776 CB ASP A 130 −25.195 34.686 2.460 1.00 71.35 C ATOM 777 CG ASP A 130 −25.925 35.902 1.916 1.00 78.10 C ATOM 778 OD1 ASP A 130 −25.369 36.570 1.008 1.00 76.18 O ATOM 779 OD2 ASP A 130 −27.049 36.181 2.399 1.00 90.58 O ATOM 780 N ARG A 131 −24.434 35.770 5.243 1.00 69.55 N ATOM 781 CA ARG A 131 −24.520 36.762 6.342 1.00 67.36 C ATOM 782 C ARG A 131 −23.143 37.268 6.778 1.00 67.63 C ATOM 783 O ARG A 131 −23.021 38.445 7.169 1.00 65.43 O ATOM 784 CB ARG A 131 −25.291 36.222 7.544 1.00 67.67 C ATOM 785 CG ARG A 131 −26.791 35.942 7.317 1.00 70.59 C ATOM 786 CD ARG A 131 −27.581 37.208 7.051 1.00 78.70 C ATOM 787 NE ARG A 131 −27.386 37.707 5.685 1.00 80.63 N ATOM 788 CZ ARG A 131 −27.690 38.934 5.244 1.00 84.09 C ATOM 789 NH1 ARG A 131 −28.227 39.861 6.051 1.00 88.30 N ATOM 790 NH2 ARG A 131 −27.454 39.237 3.969 1.00 81.21 N ATOM 791 N TYR A 132 −22.112 36.414 6.716 1.00 66.25 N ATOM 792 CA TYR A 132 −20.772 36.872 7.091 1.00 66.91 C ATOM 793 C TYR A 132 −20.300 37.951 6.123 1.00 69.24 C ATOM 794 O TYR A 132 −19.816 38.995 6.553 1.00 74.15 O ATOM 795 CB TYR A 132 −19.744 35.746 7.153 1.00 64.41 C ATOM 796 CG TYR A 132 −18.362 36.288 7.466 1.00 65.53 C ATOM 797 CD1 TYR A 132 −17.989 36.601 8.780 1.00 73.29 C ATOM 798 CD2 TYR A 132 −17.433 36.492 6.460 1.00 66.44 C ATOM 799 CE1 TYR A 132 −16.716 37.102 9.068 1.00 69.45 C ATOM 800 CE2 TYR A 132 −16.170 36.989 6.731 1.00 66.23 C ATOM 801 CZ TYR A 132 −15.809 37.293 8.025 1.00 71.49 C ATOM 802 OH TYR A 132 −14.542 37.787 8.266 1.00 69.82 O ATOM 803 N PHE A 133 −20.435 37.715 4.827 1.00 69.93 N ATOM 804 CA PHE A 133 −20.010 38.759 3.860 1.00 70.80 C ATOM 805 C PHE A 133 −20.920 39.992 3.920 1.00 69.98 C ATOM 806 O PHE A 133 −20.427 41.103 3.794 1.00 70.63 O ATOM 807 CB PHE A 133 −19.846 38.184 2.456 1.00 67.77 C ATOM 808 CG PHE A 133 −18.650 37.278 2.342 1.00 69.98 C ATOM 809 CD1 PHE A 133 −17.352 37.802 2.445 1.00 72.82 C ATOM 810 CD2 PHE A 133 −18.799 35.914 2.140 1.00 69.69 C ATOM 811 CE1 PHE A 133 −16.228 36.981 2.345 1.00 70.61 C ATOM 812 CE2 PHE A 133 −17.667 35.081 2.039 1.00 71.02 C ATOM 813 CZ PHE A 133 −16.385 35.620 2.143 1.00 68.76 C ATOM 814 N ALA A 134 −22.221 39.790 4.120 1.00 69.44 N ATOM 815 CA ALA A 134 −23.141 40.911 4.273 1.00 68.56 C ATOM 816 C ALA A 134 −22.724 41.816 5.416 1.00 68.68 C ATOM 817 O ALA A 134 −22.633 43.022 5.229 1.00 71.94 O ATOM 818 CB ALA A 134 −24.562 40.422 4.491 1.00 67.16 C ATOM 819 N ILE A 135 −22.465 41.241 6.589 1.00 68.84 N ATOM 820 CA ILE A 135 −22.145 42.039 7.792 1.00 70.46 C ATOM 821 C ILE A 135 −20.732 42.639 7.824 1.00 73.76 C ATOM 822 O ILE A 135 −20.483 43.567 8.604 1.00 77.85 O ATOM 823 CB ILE A 135 −22.361 41.205 9.107 1.00 68.09 C ATOM 824 CG1 ILE A 135 −22.694 42.122 10.290 1.00 70.69 C ATOM 825 CG2 ILE A 135 −21.151 40.315 9.387 1.00 54.90 C ATOM 826 CD1 ILE A 135 −23.130 41.371 11.531 1.00 69.39 C ATOM 827 N THR A 136 −19.819 42.123 6.998 1.00 72.13 N ATOM 828 CA THR A 136 −18.450 42.658 6.911 1.00 72.48 C ATOM 829 C THR A 136 −18.272 43.602 5.722 1.00 73.00 C ATOM 830 O THR A 136 −17.205 44.194 5.560 1.00 76.38 O ATOM 831 CB THR A 136 −17.408 41.523 6.791 1.00 71.33 C ATOM 832 OG1 THR A 136 −17.656 40.755 5.612 1.00 69.78 O ATOM 833 CG2 THR A 136 −17.478 40.622 7.980 1.00 76.96 C ATOM 834 N SER A 137 −19.312 43.743 4.890 1.00 73.57 N ATOM 835 CA SER A 137 −19.224 44.546 3.673 1.00 71.50 C ATOM 836 C SER A 137 −19.370 46.004 4.020 1.00 70.30 C ATOM 837 O SER A 137 −20.079 46.302 4.923 1.00 72.29 O ATOM 838 CB SER A 137 −20.327 44.159 2.678 1.00 70.57 C ATOM 839 OG SER A 137 −20.402 45.088 1.612 1.00 73.10 O ATOM 840 N PRO A 138 −18.693 46.924 3.292 1.00 73.40 N ATOM 841 CA PRO A 138 −18.908 48.362 3.552 1.00 71.10 C ATOM 842 C PRO A 138 −20.342 48.823 3.196 1.00 72.24 C ATOM 843 O PRO A 138 −20.791 49.845 3.689 1.00 73.27 O ATOM 844 CB PRO A 138 −17.875 49.042 2.656 1.00 72.76 C ATOM 845 CG PRO A 138 −16.952 47.966 2.220 1.00 75.11 C ATOM 846 CD PRO A 138 −17.713 46.719 2.207 1.00 70.99 C ATOM 847 N PHE A 139 −21.030 48.053 2.343 1.00 75.02 N ATOM 848 CA PHE A 139 −22.436 48.228 2.012 1.00 75.94 C ATOM 849 C PHE A 139 −23.097 47.194 2.924 1.00 81.11 C ATOM 850 O PHE A 139 −23.703 46.209 2.451 1.00 82.84 O ATOM 851 CB PHE A 139 −22.686 47.948 0.517 1.00 70.64 C ATOM 852 CG PHE A 139 −21.537 48.366 −0.376 1.00 68.22 C ATOM 853 CD1 PHE A 139 −20.705 47.417 −0.943 1.00 64.99 C ATOM 854 CD2 PHE A 139 −21.291 49.686 −0.634 1.00 54.92 C ATOM 855 CE1 PHE A 139 −19.676 47.789 −1.736 1.00 53.34 C ATOM 856 CE2 PHE A 139 −20.261 50.061 −1.427 1.00 55.02 C ATOM 857 CZ PHE A 139 −19.448 49.112 −1.982 1.00 59.66 C ATOM 858 N LYS A 140 −22.970 47.430 4.239 1.00 82.78 N ATOM 859 CA LYS A 140 −23.326 46.437 5.288 1.00 86.29 C ATOM 860 C LYS A 140 −24.732 45.890 5.161 1.00 84.99 C ATOM 861 O LYS A 140 −25.668 46.622 4.835 1.00 84.00 O ATOM 862 CB LYS A 140 −23.144 47.003 6.710 1.00 86.05 C ATOM 863 CG LYS A 140 −21.716 47.263 7.104 1.00 93.56 C ATOM 864 CD LYS A 140 −21.533 47.754 8.540 1.00 92.32 C ATOM 865 CE LYS A 140 −20.068 48.178 8.748 1.00 94.63 C ATOM 866 NZ LYS A 140 −19.803 48.731 10.115 1.00 103.84 N ATOM 867 N TYR A 141 −24.836 44.589 5.424 1.00 85.69 N ATOM 868 CA TYR A 141 −26.070 43.783 5.383 1.00 87.34 C ATOM 869 C TYR A 141 −26.642 43.451 3.984 1.00 87.45 C ATOM 870 O TYR A 141 −27.393 42.477 3.859 1.00 87.44 O ATOM 871 CB TYR A 141 −27.154 44.369 6.294 1.00 89.48 C ATOM 872 CG TYR A 141 −26.773 44.254 7.729 1.00 86.02 C ATOM 873 CD1 TYR A 141 −27.055 43.095 8.434 1.00 82.79 C ATOM 874 CD2 TYR A 141 −26.132 45.293 8.380 1.00 87.79 C ATOM 875 CE1 TYR A 141 −26.717 42.968 9.747 1.00 95.42 C ATOM 876 CE2 TYR A 141 −25.777 45.189 9.716 1.00 94.16 C ATOM 877 CZ TYR A 141 −26.071 44.020 10.400 1.00 97.24 C ATOM 878 OH TYR A 141 −25.728 43.897 11.720 1.00 91.49 O ATOM 879 N GLN A 142 −26.313 44.220 2.958 1.00 83.88 N ATOM 880 CA GLN A 142 −26.686 43.826 1.640 1.00 87.53 C ATOM 881 C GLN A 142 −25.823 42.632 1.368 1.00 85.35 C ATOM 882 O GLN A 142 −24.602 42.682 1.609 1.00 85.83 O ATOM 883 CB GLN A 142 −26.375 44.883 0.590 1.00 86.73 C ATOM 884 CG GLN A 142 −27.186 46.134 0.674 1.00 87.65 C ATOM 885 CD GLN A 142 −26.769 47.145 −0.388 1.00 91.57 C ATOM 886 OE1 GLN A 142 −26.382 46.780 −1.508 1.00 90.57 O ATOM 887 NE2 GLN A 142 −26.847 48.425 −0.040 1.00 99.82 N ATOM 888 N SER A 143 −26.446 41.562 0.879 1.00 81.37 N ATOM 889 CA SER A 143 −25.716 40.420 0.415 1.00 78.53 C ATOM 890 C SER A 143 −24.884 40.866 −0.772 1.00 77.76 C ATOM 891 O SER A 143 −25.320 41.731 −1.554 1.00 76.92 O ATOM 892 CB SER A 143 −26.662 39.315 −0.030 1.00 79.11 C ATOM 893 OG SER A 143 −25.960 38.321 −0.743 1.00 79.05 O ATOM 894 N LEU A 144 −23.695 40.284 −0.913 1.00 75.59 N ATOM 895 CA LEU A 144 −22.854 40.541 −2.082 1.00 78.96 C ATOM 896 C LEU A 144 −23.461 39.937 −3.371 1.00 77.36 C ATOM 897 O LEU A 144 −23.012 40.278 −4.477 1.00 79.84 O ATOM 898 CB LEU A 144 −21.433 40.007 −1.867 1.00 80.08 C ATOM 899 CG LEU A 144 −20.660 40.517 −0.639 1.00 82.77 C ATOM 900 CD1 LEU A 144 −19.218 40.045 −0.740 1.00 84.51 C ATOM 901 CD2 LEU A 144 −20.714 42.055 −0.474 1.00 77.04 C ATOM 902 N LEU A 145 −24.466 39.056 −3.210 1.00 74.06 N ATOM 903 CA LEU A 145 −25.162 38.394 −4.293 1.00 75.14 C ATOM 904 C LEU A 145 −26.417 39.116 −4.759 1.00 74.57 C ATOM 905 O LEU A 145 −27.158 39.675 −3.951 1.00 76.73 O ATOM 906 CB LEU A 145 −25.610 36.999 −3.836 1.00 74.59 C ATOM 907 CG LEU A 145 −24.572 36.016 −3.326 1.00 73.05 C ATOM 908 CD1 LEU A 145 −25.249 34.715 −2.973 1.00 75.64 C ATOM 909 CD2 LEU A 145 −23.502 35.785 −4.364 1.00 79.94 C ATOM 910 N THR A 146 −26.645 39.095 −6.070 1.00 74.52 N ATOM 911 CA THR A 146 −27.901 39.539 −6.654 1.00 76.04 C ATOM 912 C THR A 146 −28.928 38.413 −6.482 1.00 75.70 C ATOM 913 O THR A 146 −28.574 37.282 −6.104 1.00 75.21 O ATOM 914 CB THR A 146 −27.772 39.850 −8.156 1.00 76.46 C ATOM 915 OG1 THR A 146 −27.440 38.650 −8.860 1.00 79.82 O ATOM 916 CG2 THR A 146 −26.702 40.917 −8.423 1.00 71.60 C ATOM 917 N LYS A 147 −30.193 38.719 −6.760 1.00 77.16 N ATOM 918 CA LYS A 147 −31.279 37.718 −6.687 1.00 77.11 C ATOM 919 C LYS A 147 −30.999 36.539 −7.633 1.00 76.19 C ATOM 920 O LYS A 147 −31.219 35.383 −7.267 1.00 73.96 O ATOM 921 CB LYS A 147 −32.646 38.315 −7.052 1.00 78.39 C ATOM 922 CG LYS A 147 −33.107 39.543 −6.264 1.00 81.83 C ATOM 923 CD LYS A 147 −33.377 39.263 −4.815 1.00 87.01 C ATOM 924 CE LYS A 147 −33.758 40.559 −4.072 1.00 89.91 C ATOM 925 NZ LYS A 147 −35.048 41.155 −4.556 1.00 91.41 N ATOM 926 N ASN A 148 −30.512 36.843 −8.843 1.00 76.47 N ATOM 927 CA ASN A 148 −30.199 35.809 −9.849 1.00 77.14 C ATOM 928 C ASN A 148 −29.023 34.912 −9.476 1.00 76.20 C ATOM 929 O ASN A 148 −29.113 33.689 −9.647 1.00 74.96 O ATOM 930 CB ASN A 148 −29.958 36.431 −11.244 1.00 79.53 C ATOM 931 CG ASN A 148 −31.254 36.824 −11.956 1.00 84.32 C ATOM 932 OD1 ASN A 148 −32.355 36.395 −11.586 1.00 89.19 O ATOM 933 ND2 ASN A 148 −31.121 37.648 −12.993 1.00 85.88 N ATOM 934 N LYS A 149 −27.933 35.503 −8.976 1.00 73.79 N ATOM 935 CA LYS A 149 −26.801 34.706 −8.474 1.00 76.59 C ATOM 936 C LYS A 149 −27.254 33.809 −7.319 1.00 76.35 C ATOM 937 O LYS A 149 −26.822 32.672 −7.232 1.00 81.05 O ATOM 938 CB LYS A 149 −25.662 35.562 −7.950 1.00 79.46 C ATOM 939 CG LYS A 149 −25.007 36.526 −8.914 1.00 82.58 C ATOM 940 CD LYS A 149 −24.001 37.409 −8.129 1.00 83.85 C ATOM 941 CE LYS A 149 −23.690 38.747 −8.790 1.00 89.02 C ATOM 942 NZ LYS A 149 −23.014 39.645 −7.773 1.00 88.17 N ATOM 943 N ALA A 150 −28.120 34.331 −6.441 1.00 73.36 N ATOM 944 CA ALA A 150 −28.684 33.545 −5.325 1.00 74.46 C ATOM 945 C ALA A 150 −29.457 32.309 −5.827 1.00 75.42 C ATOM 946 O ALA A 150 −29.337 31.239 −5.245 1.00 79.11 O ATOM 947 CB ALA A 150 −29.572 34.416 −4.440 1.00 68.50 C ATOM 948 N ARG A 151 −30.243 32.465 −6.900 1.00 77.55 N ATOM 949 CA ARG A 151 −30.945 31.314 −7.511 1.00 77.51 C ATOM 950 C ARG A 151 −29.983 30.249 −8.022 1.00 77.61 C ATOM 951 O ARG A 151 −30.272 29.057 −7.913 1.00 77.15 O ATOM 952 CB ARG A 151 −31.836 31.709 −8.690 1.00 83.00 C ATOM 953 CG ARG A 151 −33.284 32.095 −8.376 1.00 89.58 C ATOM 954 CD ARG A 151 −33.576 33.556 −8.598 1.00 95.56 C ATOM 955 NE ARG A 151 −34.990 33.794 −8.884 1.00 93.67 N ATOM 956 CZ ARG A 151 −35.510 34.981 −9.220 1.00 98.85 C ATOM 957 NH1 ARG A 151 −34.743 36.084 −9.325 1.00 97.06 N ATOM 958 NH2 ARG A 151 −36.820 35.073 −9.458 1.00 102.25 N ATOM 959 N VAL A 152 −28.852 30.684 −8.579 1.00 77.11 N ATOM 960 CA VAL A 152 −27.825 29.767 −9.081 1.00 77.01 C ATOM 961 C VAL A 152 −27.144 29.030 −7.919 1.00 76.31 C ATOM 962 O VAL A 152 −26.878 27.845 −8.023 1.00 73.81 O ATOM 963 CB VAL A 152 −26.806 30.502 −9.987 1.00 78.53 C ATOM 964 CG1 VAL A 152 −25.612 29.617 −10.311 1.00 85.33 C ATOM 965 CG2 VAL A 152 −27.486 30.934 −11.274 1.00 75.42 C ATOM 966 N ILE A 153 −26.874 29.734 −6.824 1.00 76.01 N ATOM 967 CA ILE A 153 −26.278 29.102 −5.632 1.00 76.47 C ATOM 968 C ILE A 153 −27.235 28.053 −5.074 1.00 75.66 C ATOM 969 O ILE A 153 −26.812 26.945 −4.733 1.00 74.99 O ATOM 970 CB ILE A 153 −25.954 30.101 −4.523 1.00 75.93 C ATOM 971 CG1 ILE A 153 −24.899 31.127 −4.966 1.00 78.94 C ATOM 972 CG2 ILE A 153 −25.402 29.373 −3.320 1.00 75.34 C ATOM 973 CD1 ILE A 153 −23.488 30.564 −5.063 1.00 84.91 C ATOM 974 N ILE A 154 −28.517 28.416 −4.990 1.00 75.41 N ATOM 975 CA ILE A 154 −29.570 27.497 −4.545 1.00 75.93 C ATOM 976 C ILE A 154 −29.645 26.286 −5.461 1.00 72.61 C ATOM 977 O ILE A 154 −29.565 25.159 −5.001 1.00 70.17 O ATOM 978 CB ILE A 154 −30.943 28.192 −4.490 1.00 75.92 C ATOM 979 CG1 ILE A 154 −30.975 29.183 −3.328 1.00 79.46 C ATOM 980 CG2 ILE A 154 −32.072 27.170 −4.312 1.00 73.88 C ATOM 981 CD1 ILE A 154 −32.130 30.133 −3.371 1.00 81.26 C ATOM 982 N LEU A 155 −29.793 26.521 −6.754 1.00 75.14 N ATOM 983 CA LEU A 155 −29.826 25.411 −7.725 1.00 75.92 C ATOM 984 C LEU A 155 −28.604 24.497 −7.543 1.00 75.22 C ATOM 985 O LEU A 155 −28.744 23.279 −7.543 1.00 73.02 O ATOM 986 CB LEU A 155 −29.912 25.930 −9.158 1.00 71.84 C ATOM 987 CG LEU A 155 −29.837 24.903 −10.297 1.00 79.20 C ATOM 988 CD1 LEU A 155 −30.845 23.769 −10.095 1.00 82.92 C ATOM 989 CD2 LEU A 155 −30.038 25.585 −11.666 1.00 75.89 C ATOM 990 N MET A 156 −27.423 25.100 −7.384 1.00 76.89 N ATOM 991 CA MET A 156 −26.186 24.339 −7.184 1.00 77.43 C ATOM 992 C MET A 156 −26.181 23.568 −5.871 1.00 76.37 C ATOM 993 O MET A 156 −25.571 22.504 −5.801 1.00 78.59 O ATOM 994 CB MET A 156 −24.945 25.247 −7.281 1.00 81.89 C ATOM 995 CG MET A 156 −24.609 25.744 −8.716 1.00 83.04 C ATOM 996 SD MET A 156 −24.284 24.442 −9.942 1.00 100.97 S ATOM 997 CE MET A 156 −24.128 25.398 −11.458 1.00 89.68 C ATOM 998 N VAL A 157 −26.851 24.094 −4.841 1.00 76.00 N ATOM 999 CA VAL A 157 −27.000 23.370 −3.572 1.00 74.39 C ATOM 1000 C VAL A 157 −27.835 22.098 −3.784 1.00 74.09 C ATOM 1001 O VAL A 157 −27.447 21.022 −3.327 1.00 74.06 O ATOM 1002 CB VAL A 157 −27.624 24.250 −2.448 1.00 73.43 C ATOM 1003 CG1 VAL A 157 −28.152 23.382 −1.321 1.00 69.33 C ATOM 1004 CG2 VAL A 157 −26.613 25.269 −1.942 1.00 68.63 C ATOM 1005 N TRP A 158 −28.969 22.227 −4.472 1.00 74.10 N ATOM 1006 CA TRP A 158 −29.817 21.046 −4.777 1.00 74.40 C ATOM 1007 C TRP A 158 −29.131 20.024 −5.683 1.00 75.32 C ATOM 1008 O TRP A 158 −29.313 18.824 −5.482 1.00 78.30 O ATOM 1009 CB TRP A 158 −31.188 21.469 −5.310 1.00 70.20 C ATOM 1010 CG TRP A 158 −31.961 21.950 −4.183 1.00 74.02 C ATOM 1011 CD1 TRP A 158 −32.015 23.217 −3.719 1.00 80.00 C ATOM 1012 CD2 TRP A 158 −32.803 21.173 −3.333 1.00 72.24 C ATOM 1013 NE1 TRP A 158 −32.842 23.290 −2.632 1.00 79.53 N ATOM 1014 CE2 TRP A 158 −33.342 22.050 −2.371 1.00 79.45 C ATOM 1015 CE3 TRP A 158 −33.157 19.822 −3.292 1.00 73.80 C ATOM 1016 CZ2 TRP A 158 −34.220 21.625 −1.374 1.00 71.01 C ATOM 1017 CZ3 TRP A 158 −34.026 19.396 −2.307 1.00 73.95 C ATOM 1018 CH2 TRP A 158 −34.549 20.299 −1.358 1.00 74.98 C ATOM 1019 N ILE A 159 −28.354 20.495 −6.663 1.00 76.28 N ATOM 1020 CA ILE A 159 −27.572 19.595 −7.526 1.00 75.87 C ATOM 1021 C ILE A 159 −26.509 18.840 −6.713 1.00 76.85 C ATOM 1022 O ILE A 159 −26.416 17.626 −6.830 1.00 76.71 O ATOM 1023 CB ILE A 159 −26.892 20.332 −8.696 1.00 73.88 C ATOM 1024 CG1 ILE A 159 −27.933 20.757 −9.735 1.00 75.94 C ATOM 1025 CG2 ILE A 159 −25.861 19.431 −9.354 1.00 76.05 C ATOM 1026 CD1 ILE A 159 −27.359 21.529 −10.923 1.00 71.99 C ATOM 1027 N VAL A 160 −25.725 19.564 −5.903 1.00 74.32 N ATOM 1028 CA VAL A 160 −24.672 18.947 −5.073 1.00 74.37 C ATOM 1029 C VAL A 160 −25.257 17.970 −4.047 1.00 76.12 C ATOM 1030 O VAL A 160 −24.738 16.864 −3.877 1.00 72.86 O ATOM 1031 CB VAL A 160 −23.805 20.011 −4.357 1.00 75.98 C ATOM 1032 CG1 VAL A 160 −22.910 19.378 −3.297 1.00 70.80 C ATOM 1033 CG2 VAL A 160 −22.962 20.777 −5.377 1.00 72.65 C ATOM 1034 N SER A 161 −26.332 18.388 −3.376 1.00 76.74 N ATOM 1035 CA SER A 161 −27.052 17.522 −2.436 1.00 76.33 C ATOM 1036 C SER A 161 −27.588 16.253 −3.127 1.00 80.12 C ATOM 1037 O SER A 161 −27.565 15.175 −2.540 1.00 81.12 O ATOM 1038 CB SER A 161 −28.203 18.277 −1.783 1.00 77.19 C ATOM 1039 OG SER A 161 −27.721 19.391 −1.043 1.00 76.25 O ATOM 1040 N GLY A 162 −28.067 16.399 −4.367 1.00 78.97 N ATOM 1041 CA GLY A 162 −28.559 15.268 −5.165 1.00 77.45 C ATOM 1042 C GLY A 162 −27.418 14.346 −5.559 1.00 75.17 C ATOM 1043 O GLY A 162 −27.536 13.134 −5.495 1.00 72.75 O ATOM 1044 N LEU A 163 −26.313 14.958 −5.965 1.00 76.45 N ATOM 1045 CA LEU A 163 −25.098 14.253 −6.355 1.00 78.44 C ATOM 1046 C LEU A 163 −24.517 13.473 −5.171 1.00 79.50 C ATOM 1047 O LEU A 163 −23.962 12.407 −5.356 1.00 80.46 O ATOM 1048 CB LEU A 163 −24.044 15.248 −6.908 1.00 79.15 C ATOM 1049 CG LEU A 163 −23.153 14.848 −8.081 1.00 79.51 C ATOM 1050 CD1 LEU A 163 −23.978 14.520 −9.308 1.00 75.74 C ATOM 1051 CD2 LEU A 163 −22.183 15.984 −8.386 1.00 78.31 C ATOM 1052 N THR A 164 −24.650 14.007 −3.958 1.00 82.19 N ATOM 1053 CA THR A 164 −24.150 13.328 −2.751 1.00 84.31 C ATOM 1054 C THR A 164 −25.196 12.399 −2.061 1.00 85.47 C ATOM 1055 O THR A 164 −24.814 11.585 −1.225 1.00 83.60 O ATOM 1056 CB THR A 164 −23.560 14.350 −1.758 1.00 84.63 C ATOM 1057 OG1 THR A 164 −24.500 15.405 −1.514 1.00 86.96 O ATOM 1058 CG2 THR A 164 −22.291 14.950 −2.337 1.00 82.27 C ATOM 1059 N SER A 165 −26.485 12.525 −2.415 1.00 87.63 N ATOM 1060 CA SER A 165 −27.562 11.705 −1.832 1.00 88.01 C ATOM 1061 C SER A 165 −28.057 10.621 −2.778 1.00 90.10 C ATOM 1062 O SER A 165 −28.029 9.438 −2.432 1.00 94.95 O ATOM 1063 CB SER A 165 −28.745 12.583 −1.454 1.00 92.00 C ATOM 1064 OG SER A 165 −29.324 13.166 −2.614 1.00 96.93 O ATOM 1065 N PHE A 166 −28.512 11.027 −3.967 1.00 87.60 N ATOM 1066 CA PHE A 166 −29.078 10.078 −4.951 1.00 85.74 C ATOM 1067 C PHE A 166 −28.042 9.150 −5.573 1.00 85.31 C ATOM 1068 O PHE A 166 −28.313 7.967 −5.704 1.00 85.33 O ATOM 1069 CB PHE A 166 −29.884 10.789 −6.073 1.00 85.68 C ATOM 1070 CG PHE A 166 −31.350 10.954 −5.753 1.00 84.00 C ATOM 1071 CD1 PHE A 166 −32.307 10.148 −6.362 1.00 82.71 C ATOM 1072 CD2 PHE A 166 −31.773 11.910 −4.847 1.00 78.83 C ATOM 1073 CE1 PHE A 166 −33.659 10.302 −6.066 1.00 84.17 C ATOM 1074 CE2 PHE A 166 −33.124 12.065 −4.551 1.00 79.25 C ATOM 1075 CZ PHE A 166 −34.062 11.265 −5.155 1.00 80.39 C ATOM 1076 N LEU A 167 −26.870 9.673 −5.954 1.00 83.84 N ATOM 1077 CA LEU A 167 −25.839 8.813 −6.579 1.00 83.08 C ATOM 1078 C LEU A 167 −25.510 7.568 −5.738 1.00 83.34 C ATOM 1079 O LEU A 167 −25.725 6.477 −6.235 1.00 84.70 O ATOM 1080 CB LEU A 167 −24.543 9.568 −6.947 1.00 82.52 C ATOM 1081 CG LEU A 167 −24.431 10.342 −8.250 1.00 81.28 C ATOM 1082 CD1 LEU A 167 −23.003 10.847 −8.415 1.00 76.32 C ATOM 1083 CD2 LEU A 167 −24.815 9.492 −9.427 1.00 75.15 C ATOM 1084 N PRO A 168 −25.003 7.727 −4.480 1.00 82.18 N ATOM 1085 CA PRO A 168 −24.657 6.540 −3.663 1.00 81.22 C ATOM 1086 C PRO A 168 −25.803 5.561 −3.419 1.00 80.15 C ATOM 1087 O PRO A 168 −25.578 4.352 −3.439 1.00 79.49 O ATOM 1088 CB PRO A 168 −24.226 7.145 −2.321 1.00 80.06 C ATOM 1089 CG PRO A 168 −23.858 8.515 −2.614 1.00 82.53 C ATOM 1090 CD PRO A 168 −24.720 8.968 −3.734 1.00 83.35 C ATOM 1091 N ILE A 169 −27.007 6.086 −3.191 1.00 80.34 N ATOM 1092 CA ILE A 169 −28.181 5.246 −2.931 1.00 81.83 C ATOM 1093 C ILE A 169 −28.670 4.542 −4.207 1.00 83.09 C ATOM 1094 O ILE A 169 −28.974 3.347 −4.167 1.00 85.69 O ATOM 1095 CB ILE A 169 −29.316 6.029 −2.245 1.00 80.40 C ATOM 1096 CG1 ILE A 169 −28.845 6.522 −0.876 1.00 82.52 C ATOM 1097 CG2 ILE A 169 −30.538 5.150 −2.069 1.00 80.48 C ATOM 1098 CD1 ILE A 169 −29.913 7.192 −0.059 1.00 83.50 C ATOM 1099 N GLN A 170 −28.743 5.274 −5.322 1.00 83.76 N ATOM 1100 CA GLN A 170 −29.161 4.691 −6.617 1.00 83.63 C ATOM 1101 C GLN A 170 −28.056 3.817 −7.243 1.00 83.39 C ATOM 1102 O GLN A 170 −28.374 2.864 −7.960 1.00 86.11 O ATOM 1103 CB GLN A 170 −29.606 5.780 −7.616 1.00 84.12 C ATOM 1104 CG GLN A 170 −30.766 6.740 −7.123 1.00 84.07 C ATOM 1105 CD GLN A 170 −32.159 6.134 −7.119 1.00 80.98 C ATOM 1106 OE1 GLN A 170 −32.370 5.018 −7.557 1.00 77.62 O ATOM 1107 NE2 GLN A 170 −33.123 6.895 −6.614 1.00 76.26 N ATOM 1108 N MET A 171 −26.777 4.134 −6.981 1.00 82.82 N ATOM 1109 CA MET A 171 −25.636 3.286 −7.436 1.00 83.78 C ATOM 1110 C MET A 171 −25.340 2.113 −6.493 1.00 83.40 C ATOM 1111 O MET A 171 −24.487 1.279 −6.816 1.00 79.60 O ATOM 1112 CB MET A 171 −24.336 4.104 −7.631 1.00 84.08 C ATOM 1113 CG MET A 171 −24.315 5.014 −8.854 1.00 84.82 C ATOM 1114 SD MET A 171 −24.372 4.141 −10.445 1.00 93.60 S ATOM 1115 CE MET A 171 −22.962 3.018 −10.356 1.00 86.88 C ATOM 1116 N HIS A 172 −26.032 2.056 −5.346 1.00 83.18 N ATOM 1117 CA HIS A 172 −25.909 0.954 −4.373 1.00 83.41 C ATOM 1118 C HIS A 172 −24.544 0.943 −3.621 1.00 83.06 C ATOM 1119 O HIS A 172 −24.082 −0.107 −3.168 1.00 83.35 O ATOM 1120 CB HIS A 172 −26.218 −0.415 −5.050 1.00 82.96 C ATOM 1121 CG HIS A 172 −27.640 −0.563 −5.505 1.00 83.78 C ATOM 1122 ND1 HIS A 172 −28.273 −1.780 −5.517 1.00 79.73 N ATOM 1123 CD2 HIS A 172 −28.552 0.332 −5.957 1.00 82.46 C ATOM 1124 CE1 HIS A 172 −29.509 −1.639 −5.954 1.00 77.50 C ATOM 1125 NE2 HIS A 172 −29.705 −0.364 −6.229 1.00 81.47 N ATOM 1126 N TRP A 173 −23.915 2.116 −3.495 1.00 83.44 N ATOM 1127 CA TRP A 173 −22.647 2.253 −2.758 1.00 83.86 C ATOM 1128 C TRP A 173 −22.823 2.178 −1.244 1.00 83.60 C ATOM 1129 O TRP A 173 −21.888 1.833 −0.520 1.00 83.83 O ATOM 1130 CB TRP A 173 −21.953 3.576 −3.084 1.00 83.86 C ATOM 1131 CG TRP A 173 −21.492 3.720 −4.491 1.00 84.03 C ATOM 1132 CD1 TRP A 173 −21.496 2.766 −5.475 1.00 84.91 C ATOM 1133 CD2 TRP A 173 −20.939 4.893 −5.079 1.00 82.79 C ATOM 1134 NE1 TRP A 173 −20.988 3.281 −6.631 1.00 84.59 N ATOM 1135 CE2 TRP A 173 −20.636 4.585 −6.419 1.00 81.34 C ATOM 1136 CE3 TRP A 173 −20.671 6.184 −4.602 1.00 84.75 C ATOM 1137 CZ2 TRP A 173 −20.079 5.517 −7.291 1.00 85.31 C ATOM 1138 CZ3 TRP A 173 −20.115 7.115 −5.470 1.00 84.09 C ATOM 1139 CH2 TRP A 173 −19.825 6.777 −6.799 1.00 86.15 C ATOM 1140 N TYR A 174 −24.018 2.505 −0.777 1.00 81.87 N ATOM 1141 CA TYR A 174 −24.330 2.478 0.643 1.00 82.95 C ATOM 1142 C TYR A 174 −24.461 1.103 1.261 1.00 84.39 C ATOM 1143 O TYR A 174 −24.328 0.993 2.451 1.00 85.16 O ATOM 1144 CB TYR A 174 −25.646 3.224 0.916 1.00 82.89 C ATOM 1145 CG TYR A 174 −26.913 2.473 0.522 1.00 80.98 C ATOM 1146 CD1 TYR A 174 −27.679 1.794 1.475 1.00 79.89 C ATOM 1147 CD2 TYR A 174 −27.340 2.442 −0.798 1.00 79.78 C ATOM 1148 CE1 TYR A 174 −28.838 1.110 1.111 1.00 81.50 C ATOM 1149 CE2 TYR A 174 −28.489 1.767 −1.174 1.00 80.48 C ATOM 1150 CZ TYR A 174 −29.234 1.104 −0.221 1.00 81.26 C ATOM 1151 OH TYR A 174 −30.365 0.439 −0.601 1.00 79.71 O ATOM 1152 N ARG A 175 −24.709 0.070 0.457 1.00 82.91 N ATOM 1153 CA ARG A 175 −25.158 −1.227 0.984 1.00 81.71 C ATOM 1154 C ARG A 175 −24.093 −2.047 1.678 1.00 80.96 C ATOM 1155 O ARG A 175 −22.922 −1.998 1.313 1.00 80.38 O ATOM 1156 CB ARG A 175 −25.819 −2.057 −0.136 1.00 82.53 C ATOM 1157 CG ARG A 175 −26.861 −1.242 −0.863 1.00 85.26 C ATOM 1158 CD ARG A 175 −28.068 −1.948 −1.318 1.00 83.28 C ATOM 1159 NE ARG A 175 −27.867 −2.850 −2.413 1.00 82.86 N ATOM 1160 CZ ARG A 175 −28.850 −3.346 −3.172 1.00 86.74 C ATOM 1161 NH1 ARG A 175 −30.142 −3.019 −2.963 1.00 84.23 N ATOM 1162 NH2 ARG A 175 −28.541 −4.184 −4.169 1.00 81.76 N ATOM 1163 N ALA A 176 −24.539 −2.793 2.689 1.00 81.02 N ATOM 1164 CA ALA A 176 −23.696 −3.687 3.454 1.00 81.16 C ATOM 1165 C ALA A 176 −23.664 −5.020 2.737 1.00 81.72 C ATOM 1166 O ALA A 176 −24.541 −5.308 1.920 1.00 81.33 O ATOM 1167 CB ALA A 176 −24.231 −3.853 4.854 1.00 81.01 C ATOM 1168 N THR A 177 −22.651 −5.821 3.056 1.00 81.43 N ATOM 1169 CA THR A 177 −22.421 −7.128 2.422 1.00 80.81 C ATOM 1170 C THR A 177 −22.741 −8.348 3.319 1.00 81.25 C ATOM 1171 O THR A 177 −22.430 −9.481 2.947 1.00 81.14 O ATOM 1172 CB THR A 177 −20.959 −7.215 1.944 1.00 80.88 C ATOM 1173 OG1 THR A 177 −20.080 −7.111 3.071 1.00 80.59 O ATOM 1174 CG2 THR A 177 −20.656 −6.090 0.961 1.00 80.78 C ATOM 1175 N HIS A 178 −23.358 −8.123 4.482 1.00 80.75 N ATOM 1176 CA HIS A 178 −23.719 −9.227 5.397 1.00 80.59 C ATOM 1177 C HIS A 178 −25.129 −9.723 5.047 1.00 79.62 C ATOM 1178 O HIS A 178 −25.924 −8.985 4.473 1.00 80.66 O ATOM 1179 CB HIS A 178 −23.574 −8.824 6.878 1.00 80.59 C ATOM 1180 CG HIS A 178 −24.512 −7.752 7.320 1.00 80.24 C ATOM 1181 ND1 HIS A 178 −24.229 −6.410 7.189 1.00 85.34 N ATOM 1182 CD2 HIS A 178 −25.731 −7.825 7.895 1.00 81.58 C ATOM 1183 CE1 HIS A 178 −25.238 −5.702 7.664 1.00 86.20 C ATOM 1184 NE2 HIS A 178 −26.163 −6.537 8.098 1.00 88.13 N ATOM 1185 N GLN A 179 −25.413 −10.973 5.405 1.00 79.51 N ATOM 1186 CA GLN A 179 −26.652 −11.670 4.994 1.00 79.05 C ATOM 1187 C GLN A 179 −27.968 −11.024 5.453 1.00 77.54 C ATOM 1188 O GLN A 179 −28.903 −10.937 4.667 1.00 76.07 O ATOM 1189 CB GLN A 179 −26.624 −13.140 5.467 1.00 79.94 C ATOM 1190 CG GLN A 179 −27.540 −14.079 4.668 1.00 82.48 C ATOM 1191 CD GLN A 179 −27.127 −14.218 3.201 1.00 85.58 C ATOM 1192 OE1 GLN A 179 −25.963 −14.006 2.838 1.00 84.07 O ATOM 1193 NE2 GLN A 179 −28.082 −14.578 2.355 1.00 88.48 N ATOM 1194 N GLU A 180 −28.026 −10.587 6.713 1.00 76.40 N ATOM 1195 CA GLU A 180 −29.224 −9.915 7.278 1.00 76.08 C ATOM 1196 C GLU A 180 −29.656 −8.694 6.441 1.00 74.97 C ATOM 1197 O GLU A 180 −30.854 −8.437 6.268 1.00 72.77 O ATOM 1198 CB GLU A 180 −28.957 −9.518 8.738 1.00 77.18 C ATOM 1199 CG GLU A 180 −30.098 −8.829 9.499 1.00 78.62 C ATOM 1200 CD GLU A 180 −29.685 −8.430 10.920 1.00 78.93 C ATOM 1201 OE1 GLU A 180 −30.024 −7.303 11.336 1.00 77.22 O ATOM 1202 OE2 GLU A 180 −29.022 −9.239 11.618 1.00 82.04 O ATOM 1203 N ALA A 181 −28.666 −7.962 5.929 1.00 73.84 N ATOM 1204 CA ALA A 181 −28.887 −6.814 5.061 1.00 74.58 C ATOM 1205 C ALA A 181 −29.359 −7.291 3.699 1.00 74.81 C ATOM 1206 O ALA A 181 −30.389 −6.828 3.201 1.00 74.62 O ATOM 1207 CB ALA A 181 −27.610 −6.005 4.921 1.00 72.34 C ATOM 1208 N ILE A 182 −28.592 −8.218 3.110 1.00 76.12 N ATOM 1209 CA ILE A 182 −28.905 −8.843 1.800 1.00 76.87 C ATOM 1210 C ILE A 182 −30.356 −9.336 1.743 1.00 77.84 C ATOM 1211 O ILE A 182 −31.047 −9.109 0.745 1.00 78.53 O ATOM 1212 CB ILE A 182 −27.924 −10.003 1.453 1.00 76.51 C ATOM 1213 CG1 ILE A 182 −26.515 −9.466 1.192 1.00 75.02 C ATOM 1214 CG2 ILE A 182 −28.387 −10.756 0.226 1.00 76.30 C ATOM 1215 CD1 ILE A 182 −25.478 −10.540 0.959 1.00 75.60 C ATOM 1216 N ASN A 183 −30.796 −10.000 2.817 1.00 79.14 N ATOM 1217 CA ASN A 183 −32.185 −10.442 2.958 1.00 79.97 C ATOM 1218 C ASN A 183 −33.152 −9.263 2.924 1.00 81.18 C ATOM 1219 O ASN A 183 −34.160 −9.323 2.232 1.00 81.85 O ATOM 1220 CB ASN A 183 −32.392 −11.225 4.260 1.00 79.70 C ATOM 1221 CG ASN A 183 −31.725 −12.586 4.246 1.00 78.69 C ATOM 1222 OD1 ASN A 183 −30.711 −12.800 3.586 1.00 76.53 O ATOM 1223 ND2 ASN A 183 −32.301 −13.522 4.984 1.00 78.92 N ATOM 1224 N CYS A 184 −32.835 −8.200 3.671 1.00 81.90 N ATOM 1225 CA CYS A 184 −33.675 −6.990 3.698 1.00 81.53 C ATOM 1226 C CYS A 184 −33.742 −6.278 2.348 1.00 80.40 C ATOM 1227 O CYS A 184 −34.791 −5.743 1.982 1.00 80.62 O ATOM 1228 CB CYS A 184 −33.189 −5.995 4.751 1.00 82.03 C ATOM 1229 SG CYS A 184 −34.281 −4.556 4.896 1.00 86.09 S ATOM 1230 N TYR A 185 −32.630 −6.270 1.614 1.00 80.51 N ATOM 1231 CA TYR A 185 −32.588 −5.626 0.289 1.00 80.30 C ATOM 1232 C TYR A 185 −33.500 −6.328 −0.693 1.00 80.59 C ATOM 1233 O TYR A 185 −34.322 −5.683 −1.354 1.00 82.14 O ATOM 1234 CB TYR A 185 −31.165 −5.597 −0.278 1.00 79.80 C ATOM 1235 CG TYR A 185 −30.177 −4.798 0.552 1.00 80.24 C ATOM 1236 CD1 TYR A 185 −30.497 −3.522 1.018 1.00 76.86 C ATOM 1237 CD2 TYR A 185 −28.923 −5.313 0.866 1.00 79.73 C ATOM 1238 CE1 TYR A 185 −29.604 −2.797 1.769 1.00 78.10 C ATOM 1239 CE2 TYR A 185 −28.021 −4.589 1.618 1.00 79.03 C ATOM 1240 CZ TYR A 185 −28.365 −3.334 2.066 1.00 78.33 C ATOM 1241 OH TYR A 185 −27.471 −2.621 2.811 1.00 79.23 O ATOM 1242 N ALA A 186 −33.344 −7.645 −0.776 1.00 79.03 N ATOM 1243 CA ALA A 186 −34.189 −8.489 −1.622 1.00 78.43 C ATOM 1244 C ALA A 186 −35.659 −8.474 −1.178 1.00 78.71 C ATOM 1245 O ALA A 186 −36.557 −8.506 −2.023 1.00 79.49 O ATOM 1246 CB ALA A 186 −33.661 −9.914 −1.627 1.00 76.05 C ATOM 1247 N GLU A 187 −35.887 −8.422 0.140 1.00 79.25 N ATOM 1248 CA GLU A 187 −37.237 −8.399 0.713 1.00 79.62 C ATOM 1249 C GLU A 187 −37.976 −7.085 0.400 1.00 79.69 C ATOM 1250 O GLU A 187 −37.549 −5.998 0.814 1.00 80.47 O ATOM 1251 CB GLU A 187 −37.181 −8.643 2.234 1.00 79.95 C ATOM 1252 CG GLU A 187 −38.523 −8.617 2.992 1.00 82.26 C ATOM 1253 CD GLU A 187 −39.521 −9.671 2.533 1.00 86.24 C ATOM 1254 OE1 GLU A 187 −39.137 −10.643 1.845 1.00 92.89 O ATOM 1255 OE2 GLU A 187 −40.711 −9.530 2.863 1.00 88.95 O ATOM 1256 N GLU A 188 −39.081 −7.216 −0.334 1.00 79.07 N ATOM 1257 CA GLU A 188 −39.930 −6.085 −0.751 1.00 79.55 C ATOM 1258 C GLU A 188 −40.619 −5.361 0.404 1.00 79.89 C ATOM 1259 O GLU A 188 −40.897 −4.161 0.292 1.00 79.15 O ATOM 1260 CB GLU A 188 −40.983 −6.562 −1.747 1.00 79.62 C ATOM 1261 CG GLU A 188 −42.048 −7.458 −1.121 1.00 83.66 C ATOM 1262 CD GLU A 188 −42.614 −8.463 −2.084 1.00 84.99 C ATOM 1263 OE1 GLU A 188 −42.812 −8.122 −3.268 1.00 95.21 O ATOM 1264 OE2 GLU A 188 −42.866 −9.605 −1.652 1.00 89.59 O ATOM 1265 N THR A 189 −40.891 −6.089 1.496 1.00 77.96 N ATOM 1266 CA THR A 189 −41.533 −5.512 2.676 1.00 75.91 C ATOM 1267 C THR A 189 −40.541 −5.063 3.755 1.00 76.43 C ATOM 1268 O THR A 189 −40.961 −4.656 4.836 1.00 76.97 O ATOM 1269 CB THR A 189 −42.562 −6.481 3.300 1.00 75.78 C ATOM 1270 OG1 THR A 189 −41.883 −7.617 3.843 1.00 74.95 O ATOM 1271 CG2 THR A 189 −43.580 −6.923 2.252 1.00 70.98 C ATOM 1272 N CYS A 190 −39.242 −5.131 3.464 1.00 78.00 N ATOM 1273 CA CYS A 190 −38.209 −4.690 4.377 1.00 77.37 C ATOM 1274 C CYS A 190 −37.588 −3.443 3.799 1.00 74.95 C ATOM 1275 O CYS A 190 −37.131 −3.464 2.658 1.00 75.26 O ATOM 1276 CB CYS A 190 −37.136 −5.755 4.545 1.00 79.47 C ATOM 1277 SG CYS A 190 −35.924 −5.347 5.847 1.00 85.35 S ATOM 1278 N CYS A 191 −37.574 −2.368 4.582 1.00 75.05 N ATOM 1279 CA CYS A 191 −36.955 −1.110 4.175 1.00 77.17 C ATOM 1280 C CYS A 191 −36.140 −0.537 5.329 1.00 77.29 C ATOM 1281 O CYS A 191 −36.450 0.523 5.856 1.00 81.42 O ATOM 1282 CB CYS A 191 −38.024 −0.135 3.680 1.00 77.85 C ATOM 1283 SG CYS A 191 −37.383 1.370 2.899 1.00 80.61 S ATOM 1284 N ASP A 192 −35.091 −1.270 5.703 1.00 78.03 N ATOM 1285 CA ASP A 192 −34.141 −0.851 6.741 1.00 76.97 C ATOM 1286 C ASP A 192 −32.909 −0.347 6.054 1.00 75.94 C ATOM 1287 O ASP A 192 −32.428 −0.979 5.114 1.00 77.63 O ATOM 1288 CB ASP A 192 −33.762 −2.005 7.667 1.00 77.44 C ATOM 1289 CG ASP A 192 −34.889 −2.419 8.586 1.00 80.00 C ATOM 1290 OD1 ASP A 192 −35.724 −1.561 8.959 1.00 82.18 O ATOM 1291 OD2 ASP A 192 −34.941 −3.619 8.943 1.00 80.09 O ATOM 1292 N PHE A 193 −32.388 0.785 6.517 1.00 75.48 N ATOM 1293 CA PHE A 193 −31.237 1.416 5.890 1.00 75.22 C ATOM 1294 C PHE A 193 −29.909 0.865 6.443 1.00 75.43 C ATOM 1295 O PHE A 193 −29.160 1.573 7.133 1.00 73.54 O ATOM 1296 CB PHE A 193 −31.351 2.947 6.043 1.00 74.11 C ATOM 1297 CG PHE A 193 −30.399 3.730 5.175 1.00 76.00 C ATOM 1298 CD1 PHE A 193 −30.300 3.471 3.793 1.00 76.25 C ATOM 1299 CD2 PHE A 193 −29.600 4.736 5.724 1.00 76.96 C ATOM 1300 CE1 PHE A 193 −29.428 4.189 2.984 1.00 73.80 C ATOM 1301 CE2 PHE A 193 −28.718 5.465 4.905 1.00 79.72 C ATOM 1302 CZ PHE A 193 −28.638 5.183 3.528 1.00 75.51 C ATOM 1303 N PHE A 194 −29.641 −0.409 6.127 1.00 74.51 N ATOM 1304 CA PHE A 194 −28.376 −1.061 6.455 1.00 74.06 C ATOM 1305 C PHE A 194 −27.340 −0.493 5.527 1.00 74.74 C ATOM 1306 O PHE A 194 −27.542 −0.522 4.308 1.00 73.83 O ATOM 1307 CB PHE A 194 −28.423 −2.577 6.235 1.00 73.04 C ATOM 1308 CG PHE A 194 −29.226 −3.318 7.256 1.00 73.26 C ATOM 1309 CD1 PHE A 194 −28.674 −3.630 8.499 1.00 75.81 C ATOM 1310 CD2 PHE A 194 −30.534 −3.712 6.984 1.00 74.89 C ATOM 1311 CE1 PHE A 194 −29.412 −4.320 9.457 1.00 74.15 C ATOM 1312 CE2 PHE A 194 −31.282 −4.405 7.936 1.00 75.40 C ATOM 1313 CZ PHE A 194 −30.720 −4.709 9.176 1.00 74.86 C ATOM 1314 N THR A 195 −26.241 0.022 6.087 1.00 73.91 N ATOM 1315 CA THR A 195 −25.157 0.559 5.278 1.00 72.89 C ATOM 1316 C THR A 195 −23.811 −0.039 5.644 1.00 71.51 C ATOM 1317 O THR A 195 −23.636 −0.565 6.748 1.00 70.29 O ATOM 1318 CB THR A 195 −25.034 2.092 5.403 1.00 73.36 C ATOM 1319 OG1 THR A 195 −24.316 2.431 6.598 1.00 75.79 O ATOM 1320 CG2 THR A 195 −26.415 2.762 5.409 1.00 75.39 C ATOM 1321 N ASN A 196 −22.863 0.047 4.709 1.00 69.76 N ATOM 1322 CA ASN A 196 −21.483 −0.332 4.996 1.00 70.25 C ATOM 1323 C ASN A 196 −20.870 0.823 5.783 1.00 70.56 C ATOM 1324 O ASN A 196 −21.350 1.967 5.694 1.00 73.34 O ATOM 1325 CB ASN A 196 −20.676 −0.721 3.732 1.00 70.71 C ATOM 1326 CG ASN A 196 −20.478 0.426 2.743 1.00 71.43 C ATOM 1327 OD1 ASN A 196 −19.940 1.481 3.071 1.00 74.37 O ATOM 1328 ND2 ASN A 196 −20.920 0.205 1.515 1.00 72.46 N ATOM 1329 N GLN A 197 −19.824 0.524 6.544 1.00 69.71 N ATOM 1330 CA GLN A 197 −19.195 1.507 7.446 1.00 69.84 C ATOM 1331 C GLN A 197 −18.570 2.687 6.711 1.00 68.45 C ATOM 1332 O GLN A 197 −18.745 3.832 7.137 1.00 68.34 O ATOM 1333 CB GLN A 197 −18.141 0.829 8.319 1.00 70.49 C ATOM 1334 CG GLN A 197 −18.723 −0.221 9.264 1.00 73.78 C ATOM 1335 CD GLN A 197 −17.662 −1.017 9.977 1.00 75.55 C ATOM 1336 OE1 GLN A 197 −16.556 −0.528 10.223 1.00 81.19 O ATOM 1337 NE2 GLN A 197 −17.988 −2.262 10.319 1.00 83.68 N ATOM 1338 N ALA A 198 −17.855 2.400 5.617 1.00 67.03 N ATOM 1339 CA ALA A 198 −17.222 3.430 4.772 1.00 67.06 C ATOM 1340 C ALA A 198 −18.229 4.480 4.309 1.00 68.00 C ATOM 1341 O ALA A 198 −17.926 5.672 4.310 1.00 67.52 O ATOM 1342 CB ALA A 198 −16.540 2.796 3.576 1.00 67.06 C ATOM 1343 N TYR A 199 −19.420 4.027 3.916 1.00 67.41 N ATOM 1344 CA TYR A 199 −20.503 4.925 3.544 1.00 68.01 C ATOM 1345 C TYR A 199 −21.001 5.678 4.753 1.00 66.60 C ATOM 1346 O TYR A 199 −21.162 6.889 4.697 1.00 64.41 O ATOM 1347 CB TYR A 199 −21.685 4.168 2.935 1.00 69.88 C ATOM 1348 CG TYR A 199 −22.897 5.054 2.714 1.00 67.04 C ATOM 1349 CD1 TYR A 199 −23.009 5.831 1.566 1.00 70.50 C ATOM 1350 CD2 TYR A 199 −23.932 5.113 3.655 1.00 69.18 C ATOM 1351 CE1 TYR A 199 −24.128 6.657 1.352 1.00 71.51 C ATOM 1352 CE2 TYR A 199 −25.051 5.933 3.453 1.00 72.40 C ATOM 1353 CZ TYR A 199 −25.141 6.702 2.298 1.00 71.80 C ATOM 1354 OH TYR A 199 −26.236 7.509 2.088 1.00 73.79 O ATOM 1355 N ALA A 200 −21.245 4.942 5.840 1.00 67.80 N ATOM 1356 CA ALA A 200 −21.723 5.531 7.100 1.00 66.98 C ATOM 1357 C ALA A 200 −20.840 6.697 7.533 1.00 65.35 C ATOM 1358 O ALA A 200 −21.354 7.739 7.890 1.00 62.59 O ATOM 1359 CB ALA A 200 −21.789 4.486 8.183 1.00 67.92 C ATOM 1360 N ILE A 201 −19.520 6.505 7.489 1.00 66.75 N ATOM 1361 CA ILE A 201 −18.559 7.565 7.830 1.00 69.39 C ATOM 1362 C ILE A 201 −18.514 8.680 6.785 1.00 70.99 C ATOM 1363 O ILE A 201 −18.768 9.836 7.107 1.00 71.46 O ATOM 1364 CB ILE A 201 −17.126 7.027 7.971 1.00 68.97 C ATOM 1365 CG1 ILE A 201 −16.998 6.142 9.210 1.00 70.83 C ATOM 1366 CG2 ILE A 201 −16.142 8.181 8.064 1.00 68.40 C ATOM 1367 CD1 ILE A 201 −15.610 5.530 9.383 1.00 71.18 C ATOM 1368 N ALA A 202 −18.191 8.311 5.542 1.00 71.53 N ATOM 1369 CA ALA A 202 −18.001 9.273 4.438 1.00 70.39 C ATOM 1370 C ALA A 202 −19.228 10.112 4.136 1.00 71.21 C ATOM 1371 O ALA A 202 −19.112 11.331 3.979 1.00 72.58 O ATOM 1372 CB ALA A 202 −17.558 8.555 3.177 1.00 69.73 C ATOM 1373 N SER A 203 −20.395 9.474 4.054 1.00 71.88 N ATOM 1374 CA SER A 203 −21.633 10.202 3.748 1.00 72.73 C ATOM 1375 C SER A 203 −22.030 11.160 4.861 1.00 75.38 C ATOM 1376 O SER A 203 −22.457 12.274 4.579 1.00 76.69 O ATOM 1377 CB SER A 203 −22.798 9.261 3.492 1.00 73.42 C ATOM 1378 OG SER A 203 −23.944 10.010 3.118 1.00 77.91 O ATOM 1379 N SER A 204 −21.890 10.730 6.114 1.00 72.53 N ATOM 1380 CA SER A 204 −22.266 11.570 7.250 1.00 71.77 C ATOM 1381 C SER A 204 −21.283 12.742 7.442 1.00 73.36 C ATOM 1382 O SER A 204 −21.695 13.822 7.863 1.00 73.43 O ATOM 1383 CB SER A 204 −22.428 10.728 8.525 1.00 69.52 C ATOM 1384 OG SER A 204 −21.292 9.941 8.776 1.00 76.74 O ATOM 1385 N ILE A 205 −19.998 12.537 7.137 1.00 73.22 N ATOM 1386 CA ILE A 205 −19.020 13.638 7.213 1.00 73.31 C ATOM 1387 C ILE A 205 −19.284 14.674 6.118 1.00 76.06 C ATOM 1388 O ILE A 205 −19.399 15.872 6.405 1.00 76.12 O ATOM 1389 CB ILE A 205 −17.569 13.147 7.126 1.00 72.39 C ATOM 1390 CG1 ILE A 205 −17.173 12.458 8.435 1.00 71.69 C ATOM 1391 CG2 ILE A 205 −16.625 14.303 6.876 1.00 70.67 C ATOM 1392 CD1 ILE A 205 −15.812 11.794 8.390 1.00 72.10 C ATOM 1393 N VAL A 206 −19.380 14.201 4.877 1.00 74.65 N ATOM 1394 CA VAL A 206 −19.591 15.081 3.713 1.00 77.11 C ATOM 1395 C VAL A 206 −20.972 15.757 3.665 1.00 75.89 C ATOM 1396 O VAL A 206 −21.071 16.933 3.317 1.00 75.71 O ATOM 1397 CB VAL A 206 −19.356 14.321 2.388 1.00 75.98 C ATOM 1398 CG1 VAL A 206 −19.647 15.210 1.194 1.00 77.29 C ATOM 1399 CG2 VAL A 206 −17.920 13.819 2.324 1.00 80.65 C ATOM 1400 N SER A 207 −22.023 15.025 4.009 1.00 76.66 N ATOM 1401 CA SER A 207 −23.377 15.585 3.972 1.00 77.79 C ATOM 1402 C SER A 207 −23.720 16.457 5.165 1.00 78.97 C ATOM 1403 O SER A 207 −24.437 17.437 4.995 1.00 83.06 O ATOM 1404 CB SER A 207 −24.420 14.488 3.922 1.00 78.06 C ATOM 1405 OG SER A 207 −24.231 13.633 2.814 1.00 83.89 O ATOM 1406 N PHE A 208 −23.216 16.103 6.356 1.00 79.98 N ATOM 1407 CA PHE A 208 −23.601 16.771 7.609 1.00 77.07 C ATOM 1408 C PHE A 208 −22.463 17.477 8.319 1.00 78.35 C ATOM 1409 O PHE A 208 −22.511 18.695 8.470 1.00 79.76 O ATOM 1410 CB PHE A 208 −24.254 15.753 8.552 1.00 76.98 C ATOM 1411 CG PHE A 208 −24.676 16.321 9.888 1.00 78.18 C ATOM 1412 CD1 PHE A 208 −25.792 17.141 9.984 1.00 77.60 C ATOM 1413 CD2 PHE A 208 −23.957 16.025 11.051 1.00 75.04 C ATOM 1414 CE1 PHE A 208 −26.190 17.668 11.216 1.00 77.26 C ATOM 1415 CE2 PHE A 208 −24.350 16.545 12.278 1.00 80.26 C ATOM 1416 CZ PHE A 208 −25.475 17.373 12.357 1.00 77.27 C ATOM 1417 N TYR A 209 −21.450 16.739 8.756 1.00 78.29 N ATOM 1418 CA TYR A 209 −20.386 17.351 9.593 1.00 78.96 C ATOM 1419 C TYR A 209 −19.583 18.463 8.941 1.00 78.89 C ATOM 1420 O TYR A 209 −19.222 19.422 9.625 1.00 79.13 O ATOM 1421 CB TYR A 209 −19.425 16.295 10.131 1.00 80.47 C ATOM 1422 CG TYR A 209 −20.047 15.425 11.181 1.00 82.05 C ATOM 1423 CD1 TYR A 209 −20.330 14.088 10.937 1.00 80.35 C ATOM 1424 CD2 TYR A 209 −20.360 15.948 12.440 1.00 84.46 C ATOM 1425 CE1 TYR A 209 −20.906 13.292 11.913 1.00 83.44 C ATOM 1426 CE2 TYR A 209 −20.936 15.163 13.424 1.00 85.84 C ATOM 1427 CZ TYR A 209 −21.208 13.835 13.159 1.00 85.25 C ATOM 1428 OH TYR A 209 −21.776 13.073 14.145 1.00 89.70 O ATOM 1429 N VAL A 210 −19.304 18.351 7.645 1.00 78.18 N ATOM 1430 CA VAL A 210 −18.557 19.404 6.944 1.00 77.41 C ATOM 1431 C VAL A 210 −19.350 20.740 6.944 1.00 77.38 C ATOM 1432 O VAL A 210 −18.853 21.716 7.495 1.00 80.02 O ATOM 1433 CB VAL A 210 −18.108 18.976 5.515 1.00 78.51 C ATOM 1434 CG1 VAL A 210 −17.758 20.202 4.653 1.00 73.94 C ATOM 1435 CG2 VAL A 210 −16.932 18.022 5.609 1.00 76.43 C ATOM 1436 N PRO A 211 −20.557 20.780 6.339 1.00 75.78 N ATOM 1437 CA PRO A 211 −21.304 22.033 6.379 1.00 76.70 C ATOM 1438 C PRO A 211 −21.721 22.509 7.793 1.00 78.77 C ATOM 1439 O PRO A 211 −21.917 23.707 7.967 1.00 81.95 O ATOM 1440 CB PRO A 211 −22.538 21.738 5.506 1.00 78.00 C ATOM 1441 CG PRO A 211 −22.687 20.307 5.514 1.00 77.13 C ATOM 1442 CD PRO A 211 −21.305 19.746 5.601 1.00 80.23 C ATOM 1443 N LEU A 212 −21.848 21.598 8.767 1.00 77.02 N ATOM 1444 CA LEU A 212 −22.178 21.965 10.159 1.00 75.25 C ATOM 1445 C LEU A 212 −21.034 22.723 10.815 1.00 75.40 C ATOM 1446 O LEU A 212 −21.248 23.765 11.404 1.00 76.71 O ATOM 1447 CB LEU A 212 −22.492 20.727 11.017 1.00 73.79 C ATOM 1448 CG LEU A 212 −22.759 20.906 12.537 1.00 77.60 C ATOM 1449 CD1 LEU A 212 −24.102 21.571 12.759 1.00 79.25 C ATOM 1450 CD2 LEU A 212 −22.691 19.558 13.306 1.00 70.72 C ATOM 1451 N VAL A 213 −19.826 22.183 10.711 1.00 76.14 N ATOM 1452 CA VAL A 213 −18.636 22.808 11.311 1.00 76.70 C ATOM 1453 C VAL A 213 −18.372 24.190 10.713 1.00 77.08 C ATOM 1454 O VAL A 213 −18.039 25.132 11.437 1.00 76.69 O ATOM 1455 CB VAL A 213 −17.380 21.915 11.146 1.00 79.00 C ATOM 1456 CG1 VAL A 213 −16.111 22.656 11.562 1.00 78.68 C ATOM 1457 CG2 VAL A 213 −17.534 20.627 11.955 1.00 77.70 C ATOM 1458 N ILE A 214 −18.528 24.295 9.396 1.00 74.65 N ATOM 1459 CA ILE A 214 −18.372 25.561 8.690 1.00 74.83 C ATOM 1460 C ILE A 214 −19.423 26.574 9.162 1.00 72.99 C ATOM 1461 O ILE A 214 −19.075 27.692 9.502 1.00 72.81 O ATOM 1462 CB ILE A 214 −18.480 25.384 7.144 1.00 73.17 C ATOM 1463 CG1 ILE A 214 −17.318 24.547 6.603 1.00 76.80 C ATOM 1464 CG2 ILE A 214 −18.501 26.748 6.450 1.00 71.35 C ATOM 1465 CD1 ILE A 214 −17.443 24.202 5.126 1.00 72.96 C ATOM 1466 N MET A 215 −20.692 26.159 9.171 1.00 73.55 N ATOM 1467 CA MET A 215 −21.824 26.990 9.611 1.00 75.44 C ATOM 1468 C MET A 215 −21.589 27.580 10.979 1.00 77.02 C ATOM 1469 O MET A 215 −21.690 28.787 11.150 1.00 79.77 O ATOM 1470 CB MET A 215 −23.099 26.153 9.675 1.00 74.74 C ATOM 1471 CG MET A 215 −24.374 26.874 10.124 1.00 73.35 C ATOM 1472 SD MET A 215 −25.653 25.672 10.545 1.00 82.79 S ATOM 1473 CE MET A 215 −25.102 25.185 12.188 1.00 78.98 C ATOM 1474 N VAL A 216 −21.281 26.709 11.938 1.00 73.57 N ATOM 1475 CA VAL A 216 −21.029 27.111 13.319 1.00 77.98 C ATOM 1476 C VAL A 216 −19.847 28.077 13.424 1.00 78.25 C ATOM 1477 O VAL A 216 −19.960 29.124 14.061 1.00 82.48 O ATOM 1478 CB VAL A 216 −20.760 25.888 14.249 1.00 74.95 C ATOM 1479 CG1 VAL A 216 −20.358 26.350 15.613 1.00 73.89 C ATOM 1480 CG2 VAL A 216 −21.995 24.992 14.334 1.00 72.42 C ATOM 1481 N PHE A 217 −18.724 27.724 12.802 1.00 78.36 N ATOM 1482 CA PHE A 217 −17.559 28.595 12.820 1.00 77.83 C ATOM 1483 C PHE A 217 −17.880 29.962 12.227 1.00 74.98 C ATOM 1484 O PHE A 217 −17.661 30.981 12.862 1.00 74.40 O ATOM 1485 CB PHE A 217 −16.394 27.992 12.054 1.00 80.26 C ATOM 1486 CG PHE A 217 −15.236 28.930 11.918 1.00 83.15 C ATOM 1487 CD1 PHE A 217 −14.381 29.152 12.994 1.00 87.33 C ATOM 1488 CD2 PHE A 217 −14.993 29.600 10.717 1.00 87.93 C ATOM 1489 CE1 PHE A 217 −13.296 30.029 12.881 1.00 85.31 C ATOM 1490 CE2 PHE A 217 −13.912 30.478 10.592 1.00 87.26 C ATOM 1491 CZ PHE A 217 −13.063 30.692 11.677 1.00 86.57 C ATOM 1492 N VAL A 218 −18.400 29.958 11.009 1.00 72.95 N ATOM 1493 CA VAL A 218 −18.693 31.202 10.299 1.00 72.35 C ATOM 1494 C VAL A 218 −19.739 32.039 11.007 1.00 70.18 C ATOM 1495 O VAL A 218 −19.547 33.238 11.123 1.00 72.08 O ATOM 1496 CB VAL A 218 −19.126 30.965 8.828 1.00 69.97 C ATOM 1497 CG1 VAL A 218 −19.608 32.264 8.189 1.00 63.51 C ATOM 1498 CG2 VAL A 218 −17.973 30.393 8.039 1.00 69.43 C ATOM 1499 N TYR A 219 −20.822 31.428 11.479 1.00 69.91 N ATOM 1500 CA TYR A 219 −21.867 32.204 12.154 1.00 70.79 C ATOM 1501 C TYR A 219 −21.482 32.646 13.561 1.00 72.41 C ATOM 1502 O TYR A 219 −22.135 33.532 14.094 1.00 73.85 O ATOM 1503 CB TYR A 219 −23.216 31.482 12.201 1.00 70.68 C ATOM 1504 CG TYR A 219 −24.373 32.468 12.149 1.00 72.35 C ATOM 1505 CD1 TYR A 219 −24.698 33.112 10.968 1.00 75.81 C ATOM 1506 CD2 TYR A 219 −25.136 32.755 13.263 1.00 77.93 C ATOM 1507 CE1 TYR A 219 −25.757 34.025 10.892 1.00 74.63 C ATOM 1508 CE2 TYR A 219 −26.203 33.673 13.190 1.00 70.80 C ATOM 1509 CZ TYR A 219 −26.499 34.292 12.013 1.00 79.33 C ATOM 1510 OH TYR A 219 −27.544 35.187 11.958 1.00 75.98 O ATOM 1511 N SER A 220 −20.451 32.053 14.170 1.00 71.26 N ATOM 1512 CA SER A 220 −19.982 32.579 15.456 1.00 72.46 C ATOM 1513 C SER A 220 −19.265 33.886 15.124 1.00 73.33 C ATOM 1514 O SER A 220 −19.384 34.833 15.876 1.00 71.17 O ATOM 1515 CB SER A 220 −19.110 31.593 16.240 1.00 71.03 C ATOM 1516 OG SER A 220 −17.988 31.169 15.514 1.00 78.76 O ATOM 1517 N ARG A 221 −18.539 33.924 13.990 1.00 71.00 N ATOM 1518 CA ARG A 221 −17.878 35.164 13.507 1.00 71.92 C ATOM 1519 C ARG A 221 −18.847 36.268 13.102 1.00 71.03 C ATOM 1520 O ARG A 221 −18.485 37.440 13.144 1.00 75.74 O ATOM 1521 CB ARG A 221 −16.940 34.887 12.319 1.00 73.28 C ATOM 1522 CG ARG A 221 −15.789 33.967 12.607 1.00 81.79 C ATOM 1523 CD ARG A 221 −14.737 34.643 13.460 1.00 91.32 C ATOM 1524 NE ARG A 221 −13.695 33.711 13.900 1.00 99.40 N ATOM 1525 CZ ARG A 221 −12.642 34.037 14.669 1.00 104.74 C ATOM 1526 NH1 ARG A 221 −12.463 35.296 15.107 1.00 108.58 N ATOM 1527 NH2 ARG A 221 −11.754 33.095 15.009 1.00 102.27 N ATOM 1528 N VAL A 222 −20.065 35.898 12.705 1.00 72.12 N ATOM 1529 CA VAL A 222 −21.114 36.862 12.390 1.00 70.08 C ATOM 1530 C VAL A 222 −21.544 37.555 13.680 1.00 72.52 C ATOM 1531 O VAL A 222 −21.666 38.787 13.704 1.00 74.66 O ATOM 1532 CB VAL A 222 −22.314 36.199 11.688 1.00 69.66 C ATOM 1533 CG1 VAL A 222 −23.527 37.099 11.713 1.00 70.73 C ATOM 1534 CG2 VAL A 222 −21.940 35.844 10.245 1.00 74.61 C ATOM 1535 N PHE A 223 −21.771 36.782 14.752 1.00 72.29 N ATOM 1536 CA PHE A 223 −22.115 37.388 16.054 1.00 71.42 C ATOM 1537 C PHE A 223 −21.020 38.303 16.562 1.00 69.83 C ATOM 1538 O PHE A 223 −21.324 39.411 16.970 1.00 71.03 O ATOM 1539 CB PHE A 223 −22.461 36.334 17.105 1.00 72.71 C ATOM 1540 CG PHE A 223 −23.766 35.640 16.857 1.00 69.91 C ATOM 1541 CD1 PHE A 223 −24.927 36.370 16.563 1.00 71.96 C ATOM 1542 CD2 PHE A 223 −23.850 34.266 16.920 1.00 73.93 C ATOM 1543 CE1 PHE A 223 −26.121 35.741 16.338 1.00 73.93 C ATOM 1544 CE2 PHE A 223 −25.055 33.624 16.693 1.00 76.95 C ATOM 1545 CZ PHE A 223 −26.190 34.367 16.404 1.00 78.80 C ATOM 1546 N GLN A 224 −19.766 37.854 16.534 1.00 70.23 N ATOM 1547 CA GLN A 224 −18.632 38.724 16.916 1.00 71.28 C ATOM 1548 C GLN A 224 −18.655 40.034 16.155 1.00 72.34 C ATOM 1549 O GLN A 224 −18.462 41.077 16.742 1.00 76.18 O ATOM 1550 CB GLN A 224 −17.293 38.077 16.639 1.00 71.04 C ATOM 1551 CG GLN A 224 −17.007 36.807 17.433 1.00 82.15 C ATOM 1552 CD GLN A 224 −15.565 36.308 17.276 1.00 85.53 C ATOM 1553 OE1 GLN A 224 −14.706 36.986 16.673 1.00 85.57 O ATOM 1554 NE2 GLN A 224 −15.290 35.107 17.824 1.00 87.83 N ATOM 1555 N GLU A 225 −18.892 39.969 14.839 1.00 74.23 N ATOM 1556 CA GLU A 225 −18.986 41.187 14.026 1.00 73.90 C ATOM 1557 C GLU A 225 −20.161 42.043 14.442 1.00 72.00 C ATOM 1558 O GLU A 225 −19.987 43.221 14.670 1.00 69.39 O ATOM 1559 CB GLU A 225 −19.073 40.880 12.525 1.00 75.78 C ATOM 1560 CG GLU A 225 −17.713 40.806 11.881 1.00 85.48 C ATOM 1561 CD GLU A 225 −16.990 42.134 11.934 1.00 92.43 C ATOM 1562 OE1 GLU A 225 −17.670 43.174 11.759 1.00 86.05 O ATOM 1563 OE2 GLU A 225 −15.755 42.133 12.152 1.00 98.53 O ATOM 1564 N ALA A 226 −21.336 41.439 14.537 1.00 70.43 N ATOM 1565 CA ALA A 226 −22.521 42.145 14.988 1.00 71.14 C ATOM 1566 C ALA A 226 −22.238 42.861 16.326 1.00 70.13 C ATOM 1567 O ALA A 226 −22.513 44.040 16.455 1.00 69.11 O ATOM 1568 CB ALA A 226 −23.683 41.195 15.114 1.00 69.59 C ATOM 1569 N LYS A 227 −21.686 42.139 17.298 1.00 70.92 N ATOM 1570 CA LYS A 227 −21.345 42.716 18.619 1.00 72.35 C ATOM 1571 C LYS A 227 −20.374 43.880 18.510 1.00 70.68 C ATOM 1572 O LYS A 227 −20.498 44.860 19.238 1.00 71.56 O ATOM 1573 CB LYS A 227 −20.728 41.660 19.540 1.00 72.29 C ATOM 1574 CG LYS A 227 −20.673 42.077 21.027 1.00 77.11 C ATOM 1575 CD LYS A 227 −20.272 40.913 21.948 1.00 79.33 C ATOM 1576 CE LYS A 227 −20.366 41.295 23.457 1.00 89.68 C ATOM 1577 NZ LYS A 227 −19.211 42.084 23.975 1.00 91.63 N ATOM 1578 N ARG A 228 −19.419 43.749 17.591 1.00 71.06 N ATOM 1579 CA ARG A 228 −18.380 44.749 17.349 1.00 71.67 C ATOM 1580 C ARG A 228 −18.909 46.028 16.696 1.00 66.45 C ATOM 1581 O ARG A 228 −18.234 47.031 16.714 1.00 70.70 O ATOM 1582 CB ARG A 228 −17.271 44.142 16.486 1.00 73.73 C ATOM 1583 CG ARG A 228 −15.959 44.946 16.395 1.00 80.33 C ATOM 1584 CD ARG A 228 −15.060 44.487 15.239 1.00 85.20 C ATOM 1585 NE ARG A 228 −15.691 44.699 13.931 1.00 95.73 N ATOM 1586 CZ ARG A 228 −15.736 45.853 13.239 1.00 99.68 C ATOM 1587 NH1 ARG A 228 −15.178 46.979 13.702 1.00 98.15 N ATOM 1588 NH2 ARG A 228 −16.353 45.889 12.052 1.00 97.65 N ATOM 1589 N GLN A 229 −20.105 46.002 16.127 1.00 67.44 N ATOM 1590 CA GLN A 229 −20.718 47.217 15.562 1.00 69.07 C ATOM 1591 C GLN A 229 −21.324 48.107 16.630 1.00 68.99 C ATOM 1592 O GLN A 229 −21.550 49.299 16.388 1.00 73.07 O ATOM 1593 CB GLN A 229 −21.817 46.858 14.603 1.00 67.27 C ATOM 1594 CG GLN A 229 −21.328 46.085 13.403 1.00 79.11 C ATOM 1595 CD GLN A 229 −22.412 45.836 12.358 1.00 81.84 C ATOM 1596 OE1 GLN A 229 −22.097 45.437 11.233 1.00 98.22 O ATOM 1597 NE2 GLN A 229 −23.703 46.069 12.724 1.00 80.39 N ATOM 1598 N LEU A 230 −21.594 47.546 17.804 1.00 65.76 N ATOM 1599 CA LEU A 230 −22.180 48.318 18.887 1.00 66.37 C ATOM 1600 C LEU A 230 −21.228 49.424 19.268 1.00 62.49 C ATOM 1601 O LEU A 230 −20.068 49.184 19.517 1.00 63.24 O ATOM 1602 CB LEU A 230 −22.492 47.429 20.078 1.00 63.29 C ATOM 1603 CG LEU A 230 −23.526 46.346 19.805 1.00 63.55 C ATOM 1604 CD1 LEU A 230 −23.659 45.401 21.017 1.00 56.14 C ATOM 1605 CD2 LEU A 230 −24.867 46.998 19.398 1.00 63.01 C ATOM 1606 N ASN A 1002 −21.745 50.645 19.298 1.00 65.96 N ATOM 1607 CA ASN A 1002 −20.959 51.846 19.632 1.00 68.64 C ATOM 1608 C ASN A 1002 −21.911 52.946 20.059 1.00 68.56 C ATOM 1609 O ASN A 1002 −23.125 52.706 20.134 1.00 70.46 O ATOM 1610 CB ASN A 1002 −20.114 52.260 18.442 1.00 68.39 C ATOM 1611 CG ASN A 1002 −20.935 52.517 17.224 1.00 74.98 C ATOM 1612 OD1 ASN A 1002 −22.100 52.934 17.313 1.00 65.74 O ATOM 1613 ND2 ASN A 1002 −20.342 52.273 16.058 1.00 67.18 N ATOM 1614 N ILE A 1003 −21.390 54.139 20.330 1.00 69.49 N ATOM 1615 CA ILE A 1003 −22.224 55.203 20.901 1.00 70.87 C ATOM 1616 C ILE A 1003 −23.404 55.572 20.013 1.00 70.78 C ATOM 1617 O ILE A 1003 −24.472 55.886 20.521 1.00 74.04 O ATOM 1618 CB ILE A 1003 −21.362 56.453 21.310 1.00 74.80 C ATOM 1619 CG1 ILE A 1003 −22.172 57.390 22.229 1.00 76.63 C ATOM 1620 CG2 ILE A 1003 −20.788 57.153 20.075 1.00 68.47 C ATOM 1621 CD1 ILE A 1003 −21.370 58.560 22.737 1.00 73.42 C ATOM 1622 N PHE A 1004 −23.204 55.527 18.695 1.00 73.63 N ATOM 1623 CA PHE A 1004 −24.261 55.801 17.719 1.00 72.72 C ATOM 1624 C PHE A 1004 −25.409 54.841 17.835 1.00 73.43 C ATOM 1625 O PHE A 1004 −26.574 55.255 17.872 1.00 71.77 O ATOM 1626 CB PHE A 1004 −23.679 55.727 16.309 1.00 75.62 C ATOM 1627 CG PHE A 1004 −24.689 55.716 15.236 1.00 71.84 C ATOM 1628 CD1 PHE A 1004 −25.453 56.848 14.979 1.00 82.28 C ATOM 1629 CD2 PHE A 1004 −24.892 54.570 14.452 1.00 82.90 C ATOM 1630 CE1 PHE A 1004 −26.423 56.843 13.943 1.00 77.44 C ATOM 1631 CE2 PHE A 1004 −25.865 54.564 13.410 1.00 71.41 C ATOM 1632 CZ PHE A 1004 −26.620 55.697 13.166 1.00 70.16 C ATOM 1633 N GLU A 1005 −25.074 53.559 17.889 1.00 72.98 N ATOM 1634 CA GLU A 1005 −26.063 52.517 18.064 1.00 72.41 C ATOM 1635 C GLU A 1005 −26.733 52.619 19.416 1.00 74.97 C ATOM 1636 O GLU A 1005 −27.949 52.434 19.517 1.00 74.19 O ATOM 1637 CB GLU A 1005 −25.418 51.130 17.931 1.00 72.97 C ATOM 1638 CG GLU A 1005 −24.759 50.844 16.561 1.00 75.62 C ATOM 1639 CD GLU A 1005 −25.713 51.008 15.396 1.00 84.27 C ATOM 1640 OE1 GLU A 1005 −26.925 50.899 15.640 1.00 91.81 O ATOM 1641 OE2 GLU A 1005 −25.261 51.243 14.247 1.00 80.28 O ATOM 1642 N MET A 1006 −25.939 52.913 20.453 1.00 71.72 N ATOM 1643 CA MET A 1006 −26.467 53.101 21.783 1.00 72.95 C ATOM 1644 C MET A 1006 −27.562 54.182 21.796 1.00 73.35 C ATOM 1645 O MET A 1006 −28.639 53.963 22.356 1.00 73.69 O ATOM 1646 CB MET A 1006 −25.315 53.438 22.773 1.00 73.49 C ATOM 1647 CG MET A 1006 −25.769 53.933 24.131 1.00 78.05 C ATOM 1648 SD MET A 1006 −24.447 54.468 25.254 1.00 74.91 S ATOM 1649 CE MET A 1006 −24.293 53.083 26.396 1.00 86.03 C ATOM 1650 N LEU A 1007 −27.286 55.335 21.179 1.00 72.95 N ATOM 1651 CA LEU A 1007 −28.235 56.464 21.209 1.00 73.25 C ATOM 1652 C LEU A 1007 −29.347 56.268 20.193 1.00 74.44 C ATOM 1653 O LEU A 1007 −30.441 56.795 20.352 1.00 75.83 O ATOM 1654 CB LEU A 1007 −27.539 57.814 21.003 1.00 71.83 C ATOM 1655 CG LEU A 1007 −26.953 58.593 22.197 1.00 76.11 C ATOM 1656 CD1 LEU A 1007 −28.031 58.916 23.248 1.00 80.06 C ATOM 1657 CD2 LEU A 1007 −25.786 57.886 22.829 1.00 73.36 C ATOM 1658 N ARG A 1008 −29.069 55.514 19.149 1.00 77.47 N ATOM 1659 CA ARG A 1008 −30.095 55.137 18.183 1.00 77.36 C ATOM 1660 C ARG A 1008 −31.138 54.231 18.865 1.00 76.88 C ATOM 1661 O ARG A 1008 −32.311 54.329 18.575 1.00 75.51 O ATOM 1662 CB ARG A 1008 −29.437 54.430 17.040 1.00 72.89 C ATOM 1663 CG ARG A 1008 −30.325 54.077 15.936 1.00 83.81 C ATOM 1664 CD ARG A 1008 −29.530 53.460 14.840 1.00 80.34 C ATOM 1665 NE ARG A 1008 −30.368 52.534 14.093 1.00 81.78 N ATOM 1666 CZ ARG A 1008 −29.944 51.431 13.485 1.00 85.98 C ATOM 1667 NH1 ARG A 1008 −28.660 51.081 13.524 1.00 80.09 N ATOM 1668 NH2 ARG A 1008 −30.823 50.660 12.823 1.00 93.61 N ATOM 1669 N ILE A 1009 −30.678 53.355 19.773 1.00 78.10 N ATOM 1670 CA ILE A 1009 −31.548 52.465 20.543 1.00 75.58 C ATOM 1671 C ILE A 1009 −32.251 53.251 21.658 1.00 78.69 C ATOM 1672 O ILE A 1009 −33.455 53.134 21.826 1.00 81.42 O ATOM 1673 CB ILE A 1009 −30.736 51.274 21.155 1.00 78.60 C ATOM 1674 CG1 ILE A 1009 −30.220 50.329 20.057 1.00 70.72 C ATOM 1675 CG2 ILE A 1009 −31.568 50.493 22.202 1.00 73.84 C ATOM 1676 CD1 ILE A 1009 −28.924 49.579 20.447 1.00 68.79 C ATOM 1677 N ASP A 1010 −31.492 54.049 22.412 1.00 81.55 N ATOM 1678 CA ASP A 1010 −32.048 54.813 23.555 1.00 80.99 C ATOM 1679 C ASP A 1010 −32.892 56.045 23.199 1.00 82.41 C ATOM 1680 O ASP A 1010 −33.892 56.302 23.876 1.00 83.53 O ATOM 1681 CB ASP A 1010 −30.918 55.254 24.486 1.00 79.05 C ATOM 1682 CG ASP A 1010 −30.273 54.103 25.216 1.00 81.30 C ATOM 1683 OD1 ASP A 1010 −30.918 53.076 25.354 1.00 79.79 O ATOM 1684 OD2 ASP A 1010 −29.110 54.217 25.660 1.00 68.53 O ATOM 1685 N GLU A 1011 −32.499 56.790 22.163 1.00 81.15 N ATOM 1686 CA GLU A 1011 −33.206 58.046 21.749 1.00 82.09 C ATOM 1687 C GLU A 1011 −34.075 57.898 20.499 1.00 82.84 C ATOM 1688 O GLU A 1011 −35.124 58.541 20.397 1.00 86.08 O ATOM 1689 CB GLU A 1011 −32.189 59.197 21.548 1.00 85.07 C ATOM 1690 CG GLU A 1011 −32.748 60.535 20.987 1.00 86.91 C ATOM 1691 CD GLU A 1011 −33.834 61.166 21.857 1.00 92.06 C ATOM 1692 OE1 GLU A 1011 −33.791 60.980 23.098 1.00 92.05 O ATOM 1693 OE2 GLU A 1011 −34.736 61.855 21.291 1.00 82.39 O ATOM 1694 N GLY A 1012 −33.648 57.067 19.550 1.00 77.24 N ATOM 1695 CA GLY A 1012 −34.407 56.839 18.336 1.00 75.19 C ATOM 1696 C GLY A 1012 −33.925 57.768 17.246 1.00 74.64 C ATOM 1697 O GLY A 1012 −33.635 58.934 17.494 1.00 71.63 O ATOM 1698 N LEU A 1013 −33.841 57.245 16.032 1.00 74.33 N ATOM 1699 CA LEU A 1013 −33.402 58.024 14.892 1.00 73.75 C ATOM 1700 C LEU A 1013 −34.653 58.394 14.103 1.00 73.66 C ATOM 1701 O LEU A 1013 −35.397 57.511 13.670 1.00 72.82 O ATOM 1702 CB LEU A 1013 −32.419 57.214 14.060 1.00 73.46 C ATOM 1703 CG LEU A 1013 −31.520 58.014 13.118 1.00 77.16 C ATOM 1704 CD1 LEU A 1013 −30.400 57.161 12.611 1.00 81.09 C ATOM 1705 CD2 LEU A 1013 −32.304 58.573 11.978 1.00 75.18 C ATOM 1706 N ARG A 1014 −34.875 59.696 13.925 1.00 73.47 N ATOM 1707 CA ARG A 1014 −36.005 60.206 13.143 1.00 75.08 C ATOM 1708 C ARG A 1014 −35.492 60.915 11.894 1.00 73.46 C ATOM 1709 O ARG A 1014 −34.665 61.818 11.967 1.00 72.29 O ATOM 1710 CB ARG A 1014 −36.899 61.098 13.994 1.00 73.94 C ATOM 1711 CG ARG A 1014 −37.552 60.286 15.128 1.00 82.18 C ATOM 1712 CD ARG A 1014 −38.783 60.917 15.716 1.00 87.29 C ATOM 1713 NE ARG A 1014 −39.879 61.089 14.746 1.00 93.86 N ATOM 1714 CZ ARG A 1014 −41.129 61.469 15.053 1.00 92.93 C ATOM 1715 NH1 ARG A 1014 −41.488 61.729 16.323 1.00 95.92 N ATOM 1716 NH2 ARG A 1014 −42.038 61.591 14.080 1.00 92.74 N ATOM 1717 N LEU A 1015 −36.001 60.481 10.746 1.00 73.79 N ATOM 1718 CA LEU A 1015 −35.566 60.969 9.444 1.00 73.57 C ATOM 1719 C LEU A 1015 −36.284 62.239 9.033 1.00 74.92 C ATOM 1720 O LEU A 1015 −35.757 62.988 8.224 1.00 72.14 O ATOM 1721 CB LEU A 1015 −35.781 59.871 8.411 1.00 73.34 C ATOM 1722 CG LEU A 1015 −35.065 58.555 8.749 1.00 71.48 C ATOM 1723 CD1 LEU A 1015 −35.240 57.576 7.626 1.00 66.34 C ATOM 1724 CD2 LEU A 1015 −33.595 58.783 9.033 1.00 63.66 C ATOM 1725 N LYS A 1016 −37.480 62.472 9.593 1.00 77.95 N ATOM 1726 CA LYS A 1016 −38.274 63.678 9.327 1.00 75.82 C ATOM 1727 C LYS A 1016 −38.338 64.571 10.563 1.00 73.96 C ATOM 1728 O LYS A 1016 −38.379 64.065 11.680 1.00 71.82 O ATOM 1729 CB LYS A 1016 −39.688 63.302 8.908 1.00 75.21 C ATOM 1730 CG LYS A 1016 −39.744 62.451 7.649 1.00 79.49 C ATOM 1731 CD LYS A 1016 −41.159 62.214 7.176 1.00 77.17 C ATOM 1732 CE LYS A 1016 −41.160 61.358 5.900 1.00 81.57 C ATOM 1733 NZ LYS A 1016 −42.521 61.187 5.296 1.00 74.99 N ATOM 1734 N ILE A 1017 −38.347 65.889 10.334 1.00 73.18 N ATOM 1735 CA ILE A 1017 −38.511 66.900 11.372 1.00 72.14 C ATOM 1736 C ILE A 1017 −39.703 66.595 12.265 1.00 73.25 C ATOM 1737 O ILE A 1017 −40.764 66.207 11.774 1.00 74.28 O ATOM 1738 CB ILE A 1017 −38.705 68.320 10.765 1.00 71.53 C ATOM 1739 CG1 ILE A 1017 −37.391 68.862 10.210 1.00 74.05 C ATOM 1740 CG2 ILE A 1017 −39.204 69.308 11.788 1.00 70.34 C ATOM 1741 CD1 ILE A 1017 −37.443 70.318 9.833 1.00 73.51 C ATOM 1742 N TYR A 1018 −39.515 66.773 13.573 1.00 72.91 N ATOM 1743 CA TYR A 1018 −40.571 66.562 14.552 1.00 74.12 C ATOM 1744 C TYR A 1018 −40.391 67.540 15.700 1.00 73.99 C ATOM 1745 O TYR A 1018 −39.304 68.091 15.883 1.00 75.95 O ATOM 1746 CB TYR A 1018 −40.546 65.119 15.070 1.00 75.31 C ATOM 1747 CG TYR A 1018 −39.316 64.769 15.884 1.00 75.01 C ATOM 1748 CD1 TYR A 1018 −39.355 64.765 17.283 1.00 77.73 C ATOM 1749 CD2 TYR A 1018 −38.113 64.440 15.263 1.00 75.41 C ATOM 1750 CE1 TYR A 1018 −38.223 64.442 18.042 1.00 72.04 C ATOM 1751 CE2 TYR A 1018 −36.974 64.116 16.016 1.00 77.21 C ATOM 1752 CZ TYR A 1018 −37.042 64.120 17.403 1.00 76.64 C ATOM 1753 OH TYR A 1018 −35.923 63.800 18.142 1.00 79.46 O ATOM 1754 N LYS A 1019 −41.461 67.746 16.459 1.00 73.71 N ATOM 1755 CA LYS A 1019 −41.427 68.597 17.636 1.00 74.41 C ATOM 1756 C LYS A 1019 −41.056 67.729 18.826 1.00 75.37 C ATOM 1757 O LYS A 1019 −41.715 66.718 19.073 1.00 77.13 O ATOM 1758 CB LYS A 1019 −42.773 69.262 17.885 1.00 73.28 C ATOM 1759 CG LYS A 1019 −43.104 70.352 16.899 1.00 72.01 C ATOM 1760 CD LYS A 1019 −44.357 71.099 17.326 1.00 72.42 C ATOM 1761 CE LYS A 1019 −44.649 72.280 16.414 1.00 73.72 C ATOM 1762 NZ LYS A 1019 −45.910 72.993 16.796 1.00 71.74 N ATOM 1763 N ASP A 1020 −40.012 68.112 19.561 1.00 76.71 N ATOM 1764 CA ASP A 1020 −39.610 67.388 20.776 1.00 78.54 C ATOM 1765 C ASP A 1020 −40.629 67.615 21.913 1.00 78.53 C ATOM 1766 O ASP A 1020 −41.693 68.205 21.698 1.00 79.88 O ATOM 1767 CB ASP A 1020 −38.174 67.778 21.198 1.00 79.44 C ATOM 1768 CG ASP A 1020 −38.071 69.183 21.838 1.00 81.53 C ATOM 1769 OD1 ASP A 1020 −39.087 69.873 22.069 1.00 77.18 O ATOM 1770 OD2 ASP A 1020 −36.929 69.600 22.116 1.00 89.87 O ATOM 1771 N THR A 1021 −40.297 67.147 23.112 1.00 81.31 N ATOM 1772 CA THR A 1021 −41.163 67.303 24.299 1.00 81.40 C ATOM 1773 C THR A 1021 −41.464 68.771 24.674 1.00 81.89 C ATOM 1774 O THR A 1021 −42.562 69.085 25.152 1.00 83.48 O ATOM 1775 CB THR A 1021 −40.532 66.624 25.514 1.00 83.23 C ATOM 1776 OG1 THR A 1021 −39.212 67.149 25.716 1.00 86.46 O ATOM 1777 CG2 THR A 1021 −40.454 65.100 25.305 1.00 83.63 C ATOM 1778 N GLU A 1022 −40.488 69.653 24.447 1.00 81.25 N ATOM 1779 CA GLU A 1022 −40.617 71.091 24.720 1.00 80.42 C ATOM 1780 C GLU A 1022 −41.256 71.863 23.534 1.00 79.28 C ATOM 1781 O GLU A 1022 −41.339 73.093 23.576 1.00 79.62 O ATOM 1782 CB GLU A 1022 −39.243 71.710 25.032 1.00 81.04 C ATOM 1783 CG GLU A 1022 −38.301 70.940 26.011 1.00 82.43 C ATOM 1784 CD GLU A 1022 −38.788 70.891 27.448 1.00 92.01 C ATOM 1785 OE1 GLU A 1022 −39.749 71.611 27.806 1.00 94.63 O ATOM 1786 OE2 GLU A 1022 −38.192 70.117 28.231 1.00 97.00 O ATOM 1787 N GLY A 1023 −41.696 71.146 22.491 1.00 79.51 N ATOM 1788 CA GLY A 1023 −42.310 71.743 21.302 1.00 78.42 C ATOM 1789 C GLY A 1023 −41.324 72.232 20.244 1.00 78.89 C ATOM 1790 O GLY A 1023 −41.750 72.842 19.255 1.00 79.69 O ATOM 1791 N TYR A 1024 −40.021 71.971 20.436 1.00 77.49 N ATOM 1792 CA TYR A 1024 −38.981 72.476 19.532 1.00 76.94 C ATOM 1793 C TYR A 1024 −38.694 71.548 18.363 1.00 77.71 C ATOM 1794 O TYR A 1024 −38.686 70.321 18.524 1.00 79.61 O ATOM 1795 CB TYR A 1024 −37.663 72.699 20.269 1.00 77.27 C ATOM 1796 CG TYR A 1024 −37.711 73.692 21.400 1.00 79.68 C ATOM 1797 CD1 TYR A 1024 −38.145 74.987 21.192 1.00 81.27 C ATOM 1798 CD2 TYR A 1024 −37.313 73.337 22.684 1.00 79.56 C ATOM 1799 CE1 TYR A 1024 −38.189 75.913 22.232 1.00 83.68 C ATOM 1800 CE2 TYR A 1024 −37.353 74.253 23.736 1.00 78.83 C ATOM 1801 CZ TYR A 1024 −37.792 75.541 23.503 1.00 80.59 C ATOM 1802 OH TYR A 1024 −37.836 76.457 24.534 1.00 83.58 O ATOM 1803 N TYR A 1025 −38.452 72.145 17.191 1.00 74.53 N ATOM 1804 CA TYR A 1025 −38.176 71.387 15.969 1.00 73.53 C ATOM 1805 C TYR A 1025 −36.849 70.657 16.050 1.00 68.58 C ATOM 1806 O TYR A 1025 −35.801 71.285 16.188 1.00 69.06 O ATOM 1807 CB TYR A 1025 −38.195 72.295 14.736 1.00 71.74 C ATOM 1808 CG TYR A 1025 −39.564 72.806 14.416 1.00 70.34 C ATOM 1809 CD1 TYR A 1025 −39.924 74.104 14.717 1.00 71.87 C ATOM 1810 CD2 TYR A 1025 −40.510 71.983 13.810 1.00 71.28 C ATOM 1811 CE1 TYR A 1025 −41.186 74.577 14.423 1.00 77.57 C ATOM 1812 CE2 TYR A 1025 −41.783 72.445 13.509 1.00 70.46 C ATOM 1813 CZ TYR A 1025 −42.118 73.747 13.819 1.00 72.50 C ATOM 1814 OH TYR A 1025 −43.366 74.222 13.531 1.00 73.02 O ATOM 1815 N THR A 1026 −36.923 69.330 15.960 1.00 68.83 N ATOM 1816 CA THR A 1026 −35.783 68.431 16.129 1.00 69.83 C ATOM 1817 C THR A 1026 −35.730 67.408 15.000 1.00 71.01 C ATOM 1818 O THR A 1026 −36.752 67.153 14.338 1.00 75.69 O ATOM 1819 CB THR A 1026 −35.922 67.741 17.500 1.00 68.73 C ATOM 1820 OG1 THR A 1026 −36.033 68.752 18.502 1.00 71.36 O ATOM 1821 CG2 THR A 1026 −34.750 66.824 17.843 1.00 65.86 C ATOM 1822 N ILE A 1027 −34.552 66.825 14.769 1.00 69.80 N ATOM 1823 CA ILE A 1027 −34.397 65.788 13.737 1.00 72.31 C ATOM 1824 C ILE A 1027 −33.259 64.836 14.091 1.00 73.35 C ATOM 1825 O ILE A 1027 −32.339 65.215 14.794 1.00 74.44 O ATOM 1826 CB ILE A 1027 −34.153 66.428 12.350 1.00 71.44 C ATOM 1827 CG1 ILE A 1027 −34.482 65.443 11.224 1.00 73.60 C ATOM 1828 CG2 ILE A 1027 −32.713 66.934 12.235 1.00 69.68 C ATOM 1829 CD1 ILE A 1027 −34.540 66.100 9.855 1.00 69.11 C ATOM 1830 N GLY A 1028 −33.331 63.596 13.601 1.00 72.50 N ATOM 1831 CA GLY A 1028 −32.287 62.599 13.863 1.00 71.73 C ATOM 1832 C GLY A 1028 −32.366 62.107 15.292 1.00 71.31 C ATOM 1833 O GLY A 1028 −33.455 61.891 15.807 1.00 70.02 O ATOM 1834 N ILE A 1029 −31.202 61.935 15.918 1.00 73.01 N ATOM 1835 CA ILE A 1029 −31.090 61.460 17.295 1.00 72.52 C ATOM 1836 C ILE A 1029 −31.167 62.687 18.232 1.00 74.59 C ATOM 1837 O ILE A 1029 −30.158 63.152 18.782 1.00 77.27 O ATOM 1838 CB ILE A 1029 −29.786 60.637 17.526 1.00 72.24 C ATOM 1839 CG1 ILE A 1029 −29.696 59.446 16.569 1.00 71.48 C ATOM 1840 CG2 ILE A 1029 −29.716 60.140 18.998 1.00 70.08 C ATOM 1841 CD1 ILE A 1029 −28.290 58.898 16.365 1.00 72.58 C ATOM 1842 N GLY A 1030 −32.378 63.195 18.397 1.00 72.49 N ATOM 1843 CA GLY A 1030 −32.622 64.375 19.197 1.00 72.04 C ATOM 1844 C GLY A 1030 −31.760 65.582 18.861 1.00 73.16 C ATOM 1845 O GLY A 1030 −31.349 66.291 19.755 1.00 72.16 O ATOM 1846 N HIS A 1031 −31.480 65.821 17.574 1.00 75.22 N ATOM 1847 CA HIS A 1031 −30.705 67.007 17.192 1.00 74.36 C ATOM 1848 C HIS A 1031 −31.668 68.175 17.134 1.00 75.22 C ATOM 1849 O HIS A 1031 −32.614 68.137 16.371 1.00 78.88 O ATOM 1850 CB HIS A 1031 −29.985 66.864 15.840 1.00 76.09 C ATOM 1851 CG HIS A 1031 −29.190 68.068 15.470 1.00 74.73 C ATOM 1852 ND1 HIS A 1031 −27.828 68.140 15.644 1.00 73.37 N ATOM 1853 CD2 HIS A 1031 −29.568 69.255 14.941 1.00 70.52 C ATOM 1854 CE1 HIS A 1031 −27.397 69.320 15.234 1.00 72.05 C ATOM 1855 NE2 HIS A 1031 −28.434 70.016 14.805 1.00 73.60 N ATOM 1856 N LEU A 1032 −31.420 69.209 17.937 1.00 74.86 N ATOM 1857 CA LEU A 1032 −32.271 70.400 17.966 1.00 75.57 C ATOM 1858 C LEU A 1032 −31.871 71.334 16.840 1.00 75.37 C ATOM 1859 O LEU A 1032 −30.681 71.654 16.690 1.00 76.52 O ATOM 1860 CB LEU A 1032 −32.148 71.137 19.305 1.00 74.42 C ATOM 1861 CG LEU A 1032 −33.092 72.326 19.545 1.00 76.35 C ATOM 1862 CD1 LEU A 1032 −34.490 71.821 19.722 1.00 81.83 C ATOM 1863 CD2 LEU A 1032 −32.682 73.124 20.760 1.00 73.47 C ATOM 1864 N LEU A 1033 −32.857 71.770 16.057 1.00 74.07 N ATOM 1865 CA LEU A 1033 −32.610 72.675 14.928 1.00 73.31 C ATOM 1866 C LEU A 1033 −32.684 74.147 15.331 1.00 72.89 C ATOM 1867 O LEU A 1033 −31.789 74.937 15.003 1.00 70.82 O ATOM 1868 CB LEU A 1033 −33.595 72.374 13.787 1.00 73.71 C ATOM 1869 CG LEU A 1033 −33.380 71.032 13.099 1.00 68.93 C ATOM 1870 CD1 LEU A 1033 −34.527 70.727 12.146 1.00 71.95 C ATOM 1871 CD2 LEU A 1033 −32.023 71.025 12.372 1.00 66.34 C ATOM 1872 N THR A 1034 −33.751 74.502 16.039 1.00 73.89 N ATOM 1873 CA THR A 1034 −33.986 75.873 16.494 1.00 71.83 C ATOM 1874 C THR A 1034 −35.070 75.849 17.538 1.00 73.47 C ATOM 1875 O THR A 1034 −35.849 74.896 17.591 1.00 74.91 O ATOM 1876 CB THR A 1034 −34.439 76.792 15.325 1.00 71.67 C ATOM 1877 OG1 THR A 1034 −34.731 78.108 15.807 1.00 67.14 O ATOM 1878 CG2 THR A 1034 −35.685 76.231 14.634 1.00 67.88 C ATOM 1879 N LYS A 1035 −35.120 76.892 18.365 1.00 73.16 N ATOM 1880 CA LYS A 1035 −36.190 77.037 19.352 1.00 74.56 C ATOM 1881 C LYS A 1035 −37.402 77.824 18.786 1.00 74.05 C ATOM 1882 O LYS A 1035 −38.435 77.955 19.456 1.00 73.29 O ATOM 1883 CB LYS A 1035 −35.669 77.666 20.656 1.00 75.36 C ATOM 1884 CG LYS A 1035 −34.711 76.751 21.441 1.00 74.77 C ATOM 1885 CD LYS A 1035 −34.455 77.236 22.866 1.00 77.28 C ATOM 1886 CE LYS A 1035 −33.836 78.641 22.924 1.00 82.03 C ATOM 1887 NZ LYS A 1035 −33.484 79.060 24.331 1.00 81.98 N ATOM 1888 N SER A 1036 −37.256 78.330 17.559 1.00 73.73 N ATOM 1889 CA SER A 1036 −38.296 79.089 16.869 1.00 73.96 C ATOM 1890 C SER A 1036 −39.540 78.248 16.553 1.00 73.14 C ATOM 1891 O SER A 1036 −39.420 77.055 16.331 1.00 75.32 O ATOM 1892 CB SER A 1036 −37.713 79.649 15.561 1.00 74.91 C ATOM 1893 OG SER A 1036 −38.688 80.260 14.743 1.00 72.52 O ATOM 1894 N PRO A 1037 −40.740 78.874 16.532 1.00 73.94 N ATOM 1895 CA PRO A 1037 −41.968 78.169 16.153 1.00 73.36 C ATOM 1896 C PRO A 1037 −42.157 78.061 14.632 1.00 74.22 C ATOM 1897 O PRO A 1037 −43.072 77.365 14.184 1.00 76.00 O ATOM 1898 CB PRO A 1037 −43.054 79.058 16.732 1.00 72.94 C ATOM 1899 CG PRO A 1037 −42.497 80.415 16.588 1.00 73.18 C ATOM 1900 CD PRO A 1037 −41.032 80.284 16.861 1.00 73.91 C ATOM 1901 N SER A 1038 −41.308 78.744 13.855 1.00 73.88 N ATOM 1902 CA SER A 1038 −41.346 78.688 12.406 1.00 73.57 C ATOM 1903 C SER A 1038 −40.709 77.399 11.934 1.00 74.14 C ATOM 1904 O SER A 1038 −39.552 77.122 12.271 1.00 73.16 O ATOM 1905 CB SER A 1038 −40.594 79.872 11.797 1.00 73.20 C ATOM 1906 OG SER A 1038 −40.486 79.739 10.391 1.00 72.62 O ATOM 1907 N LEU A 1039 −41.463 76.617 11.163 1.00 72.03 N ATOM 1908 CA LEU A 1039 −40.961 75.378 10.590 1.00 72.88 C ATOM 1909 C LEU A 1039 −39.905 75.704 9.557 1.00 72.75 C ATOM 1910 O LEU A 1039 −38.836 75.100 9.543 1.00 74.58 O ATOM 1911 CB LEU A 1039 −42.092 74.581 9.944 1.00 72.16 C ATOM 1912 CG LEU A 1039 −41.717 73.267 9.243 1.00 71.92 C ATOM 1913 CD1 LEU A 1039 −41.053 72.299 10.198 1.00 68.87 C ATOM 1914 CD2 LEU A 1039 −42.951 72.627 8.601 1.00 71.51 C ATOM 1915 N ASN A 1040 −40.218 76.665 8.694 1.00 74.45 N ATOM 1916 CA ASN A 1040 −39.282 77.148 7.665 1.00 74.48 C ATOM 1917 C ASN A 1040 −37.918 77.589 8.246 1.00 74.80 C ATOM 1918 O ASN A 1040 −36.899 77.499 7.561 1.00 76.00 O ATOM 1919 CB ASN A 1040 −39.937 78.277 6.838 1.00 74.13 C ATOM 1920 CG ASN A 1040 −41.135 77.779 5.985 1.00 73.14 C ATOM 1921 OD1 ASN A 1040 −41.183 76.619 5.555 1.00 70.14 O ATOM 1922 ND2 ASN A 1040 −42.095 78.665 5.748 1.00 66.32 N ATOM 1923 N ALA A 1041 −37.918 78.058 9.500 1.00 74.86 N ATOM 1924 CA ALA A 1041 −36.689 78.402 10.228 1.00 75.02 C ATOM 1925 C ALA A 1041 −35.886 77.139 10.604 1.00 75.73 C ATOM 1926 O ALA A 1041 −34.664 77.155 10.555 1.00 75.98 O ATOM 1927 CB ALA A 1041 −37.011 79.220 11.466 1.00 73.60 C ATOM 1928 N ALA A 1042 −36.583 76.061 10.975 1.00 75.31 N ATOM 1929 CA ALA A 1042 −35.953 74.762 11.236 1.00 74.13 C ATOM 1930 C ALA A 1042 −35.329 74.212 9.956 1.00 75.51 C ATOM 1931 O ALA A 1042 −34.152 73.844 9.946 1.00 75.22 O ATOM 1932 CB ALA A 1042 −36.969 73.782 11.779 1.00 73.73 C ATOM 1933 N LYS A 1043 −36.135 74.164 8.887 1.00 75.74 N ATOM 1934 CA LYS A 1043 −35.689 73.728 7.547 1.00 76.54 C ATOM 1935 C LYS A 1043 −34.445 74.481 7.067 1.00 76.74 C ATOM 1936 O LYS A 1043 −33.526 73.869 6.530 1.00 78.09 O ATOM 1937 CB LYS A 1043 −36.823 73.893 6.527 1.00 75.43 C ATOM 1938 CG LYS A 1043 −37.946 72.879 6.697 1.00 76.23 C ATOM 1939 CD LYS A 1043 −39.136 73.199 5.806 1.00 82.58 C ATOM 1940 CE LYS A 1043 −40.094 72.010 5.671 1.00 85.26 C ATOM 1941 NZ LYS A 1043 −40.465 71.420 6.980 1.00 90.37 N ATOM 1942 N SER A 1044 −34.430 75.798 7.267 1.00 75.27 N ATOM 1943 CA SER A 1044 −33.272 76.626 6.931 1.00 75.10 C ATOM 1944 C SER A 1044 −32.023 76.175 7.694 1.00 75.62 C ATOM 1945 O SER A 1044 −30.957 76.023 7.097 1.00 77.80 O ATOM 1946 CB SER A 1044 −33.557 78.096 7.232 1.00 75.53 C ATOM 1947 OG SER A 1044 −32.465 78.910 6.848 1.00 75.67 O ATOM 1948 N GLU A 1045 −32.166 75.966 9.006 1.00 73.82 N ATOM 1949 CA GLU A 1045 −31.062 75.471 9.836 1.00 73.20 C ATOM 1950 C GLU A 1045 −30.641 74.064 9.456 1.00 71.02 C ATOM 1951 O GLU A 1045 −29.448 73.752 9.494 1.00 71.07 O ATOM 1952 CB GLU A 1045 −31.427 75.494 11.331 1.00 71.62 C ATOM 1953 CG GLU A 1045 −31.659 76.884 11.923 1.00 73.11 C ATOM 1954 CD GLU A 1045 −30.431 77.762 11.949 1.00 76.79 C ATOM 1955 OE1 GLU A 1045 −29.308 77.260 11.747 1.00 80.04 O ATOM 1956 OE2 GLU A 1045 −30.595 78.980 12.177 1.00 85.26 O ATOM 1957 N LEU A 1046 −31.615 73.224 9.100 1.00 70.49 N ATOM 1958 CA LEU A 1046 −31.342 71.839 8.710 1.00 73.29 C ATOM 1959 C LEU A 1046 −30.492 71.807 7.455 1.00 73.52 C ATOM 1960 O LEU A 1046 −29.453 71.144 7.434 1.00 74.73 O ATOM 1961 CB LEU A 1046 −32.644 71.064 8.469 1.00 75.19 C ATOM 1962 CG LEU A 1046 −32.556 69.562 8.130 1.00 73.72 C ATOM 1963 CD1 LEU A 1046 −31.881 68.775 9.243 1.00 74.36 C ATOM 1964 CD2 LEU A 1046 −33.942 69.000 7.851 1.00 73.43 C ATOM 1965 N ASP A 1047 −30.945 72.529 6.426 1.00 72.94 N ATOM 1966 CA ASP A 1047 −30.225 72.645 5.148 1.00 75.39 C ATOM 1967 C ASP A 1047 −28.812 73.161 5.343 1.00 74.85 C ATOM 1968 O ASP A 1047 −27.875 72.634 4.758 1.00 76.38 O ATOM 1969 CB ASP A 1047 −30.953 73.595 4.160 1.00 74.34 C ATOM 1970 CG ASP A 1047 −32.327 73.076 3.713 1.00 75.72 C ATOM 1971 OD1 ASP A 1047 −32.707 71.938 4.072 1.00 72.63 O ATOM 1972 OD2 ASP A 1047 −33.029 73.824 2.995 1.00 80.04 O ATOM 1973 N LYS A 1048 −28.677 74.193 6.168 1.00 75.86 N ATOM 1974 CA LYS A 1048 −27.379 74.779 6.494 1.00 75.59 C ATOM 1975 C LYS A 1048 −26.464 73.779 7.248 1.00 75.44 C ATOM 1976 O LYS A 1048 −25.241 73.810 7.088 1.00 75.09 O ATOM 1977 CB LYS A 1048 −27.603 76.061 7.296 1.00 75.61 C ATOM 1978 CG LYS A 1048 −26.368 76.896 7.555 1.00 77.43 C ATOM 1979 CD LYS A 1048 −26.729 78.377 7.838 1.00 78.34 C ATOM 1980 CE LYS A 1048 −27.671 78.567 9.048 1.00 80.49 C ATOM 1981 NZ LYS A 1048 −27.074 78.073 10.322 1.00 79.34 N ATOM 1982 N ALA A 1049 −27.073 72.903 8.055 1.00 73.58 N ATOM 1983 CA ALA A 1049 −26.345 71.870 8.804 1.00 74.82 C ATOM 1984 C ALA A 1049 −25.950 70.699 7.909 1.00 75.13 C ATOM 1985 O ALA A 1049 −24.797 70.271 7.913 1.00 75.27 O ATOM 1986 CB ALA A 1049 −27.188 71.364 9.973 1.00 73.33 C ATOM 1987 N ILE A 1050 −26.924 70.194 7.152 1.00 73.31 N ATOM 1988 CA ILE A 1050 −26.746 69.038 6.255 1.00 73.24 C ATOM 1989 C ILE A 1050 −25.994 69.378 4.951 1.00 72.49 C ATOM 1990 O ILE A 1050 −25.155 68.602 4.500 1.00 72.34 O ATOM 1991 CB ILE A 1050 −28.124 68.397 5.931 1.00 73.95 C ATOM 1992 CG1 ILE A 1050 −28.758 67.828 7.207 1.00 72.34 C ATOM 1993 CG2 ILE A 1050 −27.996 67.297 4.907 1.00 70.31 C ATOM 1994 CD1 ILE A 1050 −27.968 66.650 7.819 1.00 75.79 C ATOM 1995 N GLY A 1051 −26.304 70.530 4.362 1.00 70.45 N ATOM 1996 CA GLY A 1051 −25.628 71.022 3.152 1.00 69.29 C ATOM 1997 C GLY A 1051 −26.371 70.810 1.841 1.00 68.84 C ATOM 1998 O GLY A 1051 −25.741 70.624 0.800 1.00 64.99 O ATOM 1999 N ARG A 1052 −27.703 70.841 1.900 1.00 66.56 N ATOM 2000 CA ARG A 1052 −28.563 70.680 0.730 1.00 67.90 C ATOM 2001 C ARG A 1052 −29.995 71.012 1.090 1.00 68.30 C ATOM 2002 O ARG A 1052 −30.309 71.175 2.261 1.00 71.80 O ATOM 2003 CB ARG A 1052 −28.502 69.251 0.162 1.00 66.98 C ATOM 2004 CG ARG A 1052 −28.971 68.148 1.076 1.00 72.44 C ATOM 2005 CD ARG A 1052 −29.034 66.833 0.330 1.00 70.28 C ATOM 2006 NE ARG A 1052 −29.469 65.745 1.210 1.00 74.46 N ATOM 2007 CZ ARG A 1052 −30.740 65.449 1.540 1.00 75.81 C ATOM 2008 NH1 ARG A 1052 −31.772 66.146 1.076 1.00 75.31 N ATOM 2009 NH2 ARG A 1052 −30.991 64.430 2.354 1.00 78.34 N ATOM 2010 N ASN A 1053 −30.856 71.108 0.083 1.00 67.63 N ATOM 2011 CA ASN A 1053 −32.289 71.307 0.299 1.00 69.73 C ATOM 2012 C ASN A 1053 −32.876 69.984 0.719 1.00 69.35 C ATOM 2013 O ASN A 1053 −33.005 69.075 −0.095 1.00 69.80 O ATOM 2014 CB ASN A 1053 −32.996 71.829 −0.957 1.00 70.79 C ATOM 2015 CG ASN A 1053 −32.759 73.310 −1.201 1.00 72.54 C ATOM 2016 OD1 ASN A 1053 −33.223 73.847 −2.196 1.00 78.00 O ATOM 2017 ND2 ASN A 1053 −32.037 73.975 −0.292 1.00 81.34 N ATOM 2018 N THR A 1054 −33.228 69.880 1.996 1.00 72.38 N ATOM 2019 CA THR A 1054 −33.753 68.647 2.552 1.00 72.53 C ATOM 2020 C THR A 1054 −35.266 68.588 2.510 1.00 73.02 C ATOM 2021 O THR A 1054 −35.826 67.516 2.305 1.00 77.21 O ATOM 2022 CB THR A 1054 −33.251 68.458 3.982 1.00 71.32 C ATOM 2023 OG1 THR A 1054 −33.557 69.619 4.758 1.00 71.59 O ATOM 2024 CG2 THR A 1054 −31.748 68.254 3.981 1.00 73.10 C ATOM 2025 N ASN A 1055 −35.908 69.737 2.702 1.00 75.24 N ATOM 2026 CA ASN A 1055 −37.360 69.842 2.815 1.00 76.78 C ATOM 2027 C ASN A 1055 −37.876 69.053 4.046 1.00 76.94 C ATOM 2028 O ASN A 1055 −38.985 68.522 4.044 1.00 82.59 O ATOM 2029 CB ASN A 1055 −38.062 69.398 1.517 1.00 76.21 C ATOM 2030 CG ASN A 1055 −39.511 69.867 1.431 1.00 78.54 C ATOM 2031 OD1 ASN A 1055 −39.930 70.812 2.109 1.00 77.71 O ATOM 2032 ND2 ASN A 1055 −40.285 69.199 0.587 1.00 87.37 N ATOM 2033 N GLY A 1056 −37.054 68.984 5.091 1.00 76.49 N ATOM 2034 CA GLY A 1056 −37.403 68.283 6.314 1.00 76.60 C ATOM 2035 C GLY A 1056 −36.995 66.832 6.409 1.00 74.80 C ATOM 2036 O GLY A 1056 −37.028 66.298 7.489 1.00 77.03 O ATOM 2037 N VAL A 1057 −36.610 66.192 5.301 1.00 74.21 N ATOM 2038 CA VAL A 1057 −36.246 64.770 5.320 1.00 73.68 C ATOM 2039 C VAL A 1057 −34.745 64.554 5.095 1.00 73.15 C ATOM 2040 O VAL A 1057 −34.181 65.114 4.151 1.00 77.13 O ATOM 2041 CB VAL A 1057 −37.029 63.981 4.261 1.00 72.56 C ATOM 2042 CG1 VAL A 1057 −36.711 62.498 4.368 1.00 74.94 C ATOM 2043 CG2 VAL A 1057 −38.523 64.201 4.432 1.00 70.86 C ATOM 2044 N ILE A 1058 −34.120 63.742 5.963 1.00 71.83 N ATOM 2045 CA ILE A 1058 −32.698 63.347 5.843 1.00 71.92 C ATOM 2046 C ILE A 1058 −32.520 61.833 5.872 1.00 73.30 C ATOM 2047 O ILE A 1058 −33.382 61.111 6.370 1.00 76.64 O ATOM 2048 CB ILE A 1058 −31.815 63.953 6.956 1.00 67.96 C ATOM 2049 CG1 ILE A 1058 −32.220 63.448 8.364 1.00 71.45 C ATOM 2050 CG2 ILE A 1058 −31.883 65.457 6.899 1.00 65.81 C ATOM 2051 CD1 ILE A 1058 −31.205 63.816 9.445 1.00 65.73 C ATOM 2052 N THR A 1059 −31.392 61.362 5.343 1.00 72.25 N ATOM 2053 CA THR A 1059 −31.070 59.932 5.345 1.00 71.10 C ATOM 2054 C THR A 1059 −30.533 59.545 6.712 1.00 70.11 C ATOM 2055 O THR A 1059 −30.166 60.405 7.495 1.00 71.12 O ATOM 2056 CB THR A 1059 −29.990 59.595 4.353 1.00 70.42 C ATOM 2057 OG1 THR A 1059 −28.775 60.228 4.776 1.00 67.63 O ATOM 2058 CG2 THR A 1059 −30.377 60.056 2.874 1.00 63.19 C ATOM 2059 N LYS A 1060 −30.493 58.244 6.972 1.00 70.88 N ATOM 2060 CA LYS A 1060 −29.950 57.684 8.208 1.00 71.95 C ATOM 2061 C LYS A 1060 −28.481 58.087 8.416 1.00 72.94 C ATOM 2062 O LYS A 1060 −28.082 58.452 9.536 1.00 71.54 O ATOM 2063 CB LYS A 1060 −30.074 56.151 8.215 1.00 70.63 C ATOM 2064 CG LYS A 1060 −29.325 55.466 9.387 1.00 75.49 C ATOM 2065 CD LYS A 1060 −29.500 53.953 9.419 1.00 73.88 C ATOM 2066 CE LYS A 1060 −28.422 53.322 10.308 1.00 71.79 C ATOM 2067 NZ LYS A 1060 −28.584 51.848 10.427 1.00 80.16 N ATOM 2068 N ASP A 1061 −27.706 58.010 7.330 1.00 68.84 N ATOM 2069 CA ASP A 1061 −26.282 58.339 7.320 1.00 68.20 C ATOM 2070 C ASP A 1061 −26.043 59.814 7.586 1.00 66.58 C ATOM 2071 O ASP A 1061 −25.076 60.148 8.255 1.00 70.66 O ATOM 2072 CB ASP A 1061 −25.622 57.923 5.983 1.00 64.64 C ATOM 2073 CG ASP A 1061 −25.572 56.386 5.788 1.00 73.92 C ATOM 2074 OD1 ASP A 1061 −25.501 55.619 6.782 1.00 83.01 O ATOM 2075 OD2 ASP A 1061 −25.603 55.935 4.630 1.00 82.92 O ATOM 2076 N GLU A 1062 −26.909 60.687 7.071 1.00 60.10 N ATOM 2077 CA GLU A 1062 −26.856 62.113 7.437 1.00 62.56 C ATOM 2078 C GLU A 1062 −27.203 62.317 8.955 1.00 64.45 C ATOM 2079 O GLU A 1062 −26.559 63.127 9.619 1.00 62.28 O ATOM 2080 CB GLU A 1062 −27.770 62.910 6.572 1.00 60.74 C ATOM 2081 CG GLU A 1062 −27.243 63.018 5.142 1.00 60.93 C ATOM 2082 CD GLU A 1062 −28.230 63.606 4.198 1.00 68.90 C ATOM 2083 OE1 GLU A 1062 −29.446 63.498 4.488 1.00 67.42 O ATOM 2084 OE2 GLU A 1062 −27.795 64.182 3.155 1.00 73.08 O ATOM 2085 N ALA A 1063 −28.197 61.590 9.485 1.00 64.40 N ATOM 2086 CA ALA A 1063 −28.494 61.647 10.955 1.00 68.01 C ATOM 2087 C ALA A 1063 −27.251 61.296 11.748 1.00 69.72 C ATOM 2088 O ALA A 1063 −26.948 61.940 12.745 1.00 79.20 O ATOM 2089 CB ALA A 1063 −29.637 60.723 11.331 1.00 65.57 C ATOM 2090 N GLU A 1064 −26.529 60.269 11.298 1.00 73.91 N ATOM 2091 CA GLU A 1064 −25.261 59.873 11.920 1.00 71.50 C ATOM 2092 C GLU A 1064 −24.218 60.973 11.815 1.00 72.04 C ATOM 2093 O GLU A 1064 −23.465 61.158 12.747 1.00 76.73 O ATOM 2094 CB GLU A 1064 −24.746 58.584 11.294 1.00 70.30 C ATOM 2095 CG GLU A 1064 −23.391 58.090 11.772 1.00 71.10 C ATOM 2096 CD GLU A 1064 −23.012 56.715 11.195 1.00 81.17 C ATOM 2097 OE1 GLU A 1064 −23.711 56.194 10.251 1.00 87.16 O ATOM 2098 OE2 GLU A 1064 −22.006 56.150 11.688 1.00 89.77 O ATOM 2099 N LYS A 1065 −24.168 61.701 10.695 1.00 73.67 N ATOM 2100 CA LYS A 1065 −23.204 62.821 10.568 1.00 73.69 C ATOM 2101 C LYS A 1065 −23.466 63.903 11.620 1.00 70.10 C ATOM 2102 O LYS A 1065 −22.553 64.325 12.310 1.00 68.11 O ATOM 2103 CB LYS A 1065 −23.232 63.461 9.190 1.00 75.31 C ATOM 2104 CG LYS A 1065 −22.891 62.546 8.022 1.00 80.59 C ATOM 2105 CD LYS A 1065 −21.422 62.223 7.938 1.00 85.80 C ATOM 2106 CE LYS A 1065 −21.098 61.379 6.680 1.00 81.37 C ATOM 2107 NZ LYS A 1065 −21.661 61.944 5.425 1.00 72.55 N ATOM 2108 N LEU A 1066 −24.718 64.335 11.723 1.00 69.54 N ATOM 2109 CA LEU A 1066 −25.133 65.305 12.748 1.00 71.70 C ATOM 2110 C LEU A 1066 −24.854 64.804 14.165 1.00 70.08 C ATOM 2111 O LEU A 1066 −24.476 65.572 15.033 1.00 68.08 O ATOM 2112 CB LEU A 1066 −26.621 65.605 12.636 1.00 71.78 C ATOM 2113 CG LEU A 1066 −27.056 66.466 11.468 1.00 76.18 C ATOM 2114 CD1 LEU A 1066 −28.565 66.357 11.261 1.00 78.42 C ATOM 2115 CD2 LEU A 1066 −26.625 67.899 11.715 1.00 77.95 C ATOM 2116 N PHE A 1067 −25.049 63.513 14.373 1.00 71.17 N ATOM 2117 CA PHE A 1067 −24.844 62.899 15.673 1.00 71.75 C ATOM 2118 C PHE A 1067 −23.383 62.902 16.104 1.00 71.56 C ATOM 2119 O PHE A 1067 −23.098 63.171 17.275 1.00 76.85 O ATOM 2120 CB PHE A 1067 −25.378 61.480 15.669 1.00 74.38 C ATOM 2121 CG PHE A 1067 −25.046 60.740 16.880 1.00 75.45 C ATOM 2122 CD1 PHE A 1067 −25.810 60.897 18.012 1.00 78.29 C ATOM 2123 CD2 PHE A 1067 −23.957 59.877 16.909 1.00 81.22 C ATOM 2124 CE1 PHE A 1067 −25.509 60.221 19.138 1.00 77.84 C ATOM 2125 CE2 PHE A 1067 −23.650 59.194 18.043 1.00 81.99 C ATOM 2126 CZ PHE A 1067 −24.425 59.361 19.165 1.00 78.68 C ATOM 2127 N ASN A 1068 −22.464 62.611 15.188 1.00 72.06 N ATOM 2128 CA ASN A 1068 −21.035 62.658 15.520 1.00 72.87 C ATOM 2129 C ASN A 1068 −20.611 64.092 15.888 1.00 74.75 C ATOM 2130 O ASN A 1068 −19.722 64.272 16.716 1.00 76.41 O ATOM 2131 CB ASN A 1068 −20.134 62.151 14.400 1.00 70.30 C ATOM 2132 CG ASN A 1068 −20.477 60.741 13.936 1.00 77.74 C ATOM 2133 OD1 ASN A 1068 −20.902 59.903 14.722 1.00 71.67 O ATOM 2134 ND2 ASN A 1068 −20.286 60.479 12.617 1.00 72.67 N ATOM 2135 N GLN A 1069 −21.244 65.094 15.275 1.00 73.62 N ATOM 2136 CA GLN A 1069 −20.982 66.487 15.631 1.00 73.62 C ATOM 2137 C GLN A 1069 −21.541 66.803 17.020 1.00 73.06 C ATOM 2138 O GLN A 1069 −20.874 67.455 17.802 1.00 68.92 O ATOM 2139 CB GLN A 1069 −21.586 67.449 14.634 1.00 71.42 C ATOM 2140 CG GLN A 1069 −20.967 67.379 13.249 1.00 81.86 C ATOM 2141 CD GLN A 1069 −21.717 68.235 12.239 1.00 81.55 C ATOM 2142 OE1 GLN A 1069 −22.504 69.116 12.614 1.00 87.86 O ATOM 2143 NE2 GLN A 1069 −21.480 67.980 10.948 1.00 91.37 N ATOM 2144 N ASP A 1070 −22.759 66.341 17.304 1.00 69.64 N ATOM 2145 CA ASP A 1070 −23.362 66.549 18.615 1.00 70.15 C ATOM 2146 C ASP A 1070 −22.510 65.879 19.689 1.00 70.56 C ATOM 2147 O ASP A 1070 −22.272 66.486 20.737 1.00 71.26 O ATOM 2148 CB ASP A 1070 −24.795 66.038 18.664 1.00 68.69 C ATOM 2149 CG ASP A 1070 −25.742 66.828 17.756 1.00 77.13 C ATOM 2150 OD1 ASP A 1070 −25.444 67.997 17.392 1.00 75.90 O ATOM 2151 OD2 ASP A 1070 −26.794 66.277 17.400 1.00 74.40 O ATOM 2152 N VAL A 1071 −22.058 64.649 19.426 1.00 67.27 N ATOM 2153 CA VAL A 1071 −21.150 63.948 20.341 1.00 69.23 C ATOM 2154 C VAL A 1071 −19.906 64.780 20.577 1.00 70.51 C ATOM 2155 O VAL A 1071 −19.509 64.968 21.714 1.00 71.56 O ATOM 2156 CB VAL A 1071 −20.737 62.563 19.820 1.00 66.16 C ATOM 2157 CG1 VAL A 1071 −19.561 62.086 20.548 1.00 63.68 C ATOM 2158 CG2 VAL A 1071 −21.887 61.570 19.952 1.00 68.51 C ATOM 2159 N ASP A 1072 −19.294 65.280 19.500 1.00 74.44 N ATOM 2160 CA ASP A 1072 −18.128 66.154 19.623 1.00 73.07 C ATOM 2161 C ASP A 1072 −18.410 67.321 20.555 1.00 74.85 C ATOM 2162 O ASP A 1072 −17.628 67.585 21.464 1.00 78.47 O ATOM 2163 CB ASP A 1072 −17.688 66.703 18.269 1.00 77.99 C ATOM 2164 CG ASP A 1072 −16.502 67.645 18.387 1.00 82.49 C ATOM 2165 OD1 ASP A 1072 −15.355 67.156 18.521 1.00 93.79 O ATOM 2166 OD2 ASP A 1072 −16.730 68.874 18.341 1.00 88.07 O ATOM 2167 N ALA A 1073 −19.530 68.003 20.316 1.00 70.34 N ATOM 2168 CA ALA A 1073 −19.981 69.126 21.153 1.00 71.13 C ATOM 2169 C ALA A 1073 −20.178 68.742 22.643 1.00 72.31 C ATOM 2170 O ALA A 1073 −19.771 69.495 23.520 1.00 72.64 O ATOM 2171 CB ALA A 1073 −21.261 69.675 20.620 1.00 68.05 C ATOM 2172 N ALA A 1074 −20.797 67.584 22.890 1.00 69.54 N ATOM 2173 CA ALA A 1074 −21.085 67.096 24.244 1.00 71.93 C ATOM 2174 C ALA A 1074 −19.795 66.903 25.028 1.00 72.36 C ATOM 2175 O ALA A 1074 −19.684 67.378 26.149 1.00 77.33 O ATOM 2176 CB ALA A 1074 −21.851 65.807 24.193 1.00 71.13 C ATOM 2177 N VAL A 1075 −18.836 66.207 24.415 1.00 72.46 N ATOM 2178 CA VAL A 1075 −17.511 65.973 24.995 1.00 74.25 C ATOM 2179 C VAL A 1075 −16.752 67.262 25.348 1.00 74.70 C ATOM 2180 O VAL A 1075 −16.163 67.345 26.429 1.00 74.40 O ATOM 2181 CB VAL A 1075 −16.648 65.102 24.073 1.00 74.74 C ATOM 2182 CG1 VAL A 1075 −15.208 65.076 24.539 1.00 75.33 C ATOM 2183 CG2 VAL A 1075 −17.205 63.696 24.033 1.00 76.41 C ATOM 2184 N ARG A 1076 −16.768 68.246 24.455 1.00 72.53 N ATOM 2185 CA ARG A 1076 −16.124 69.546 24.726 1.00 74.21 C ATOM 2186 C ARG A 1076 −16.766 70.230 25.911 1.00 74.53 C ATOM 2187 O ARG A 1076 −16.070 70.838 26.735 1.00 74.38 O ATOM 2188 CB ARG A 1076 −16.204 70.487 23.533 1.00 70.53 C ATOM 2189 CG ARG A 1076 −15.356 70.070 22.367 1.00 76.54 C ATOM 2190 CD ARG A 1076 −15.465 71.070 21.205 1.00 82.85 C ATOM 2191 NE ARG A 1076 −15.065 70.430 19.951 1.00 91.84 N ATOM 2192 CZ ARG A 1076 −13.802 70.183 19.569 1.00 96.99 C ATOM 2193 NH1 ARG A 1076 −12.746 70.517 20.334 1.00 97.76 N ATOM 2194 NH2 ARG A 1076 −13.580 69.588 18.393 1.00 103.37 N ATOM 2195 N GLY A 1077 −18.091 70.120 25.977 1.00 72.90 N ATOM 2196 CA GLY A 1077 −18.865 70.656 27.059 1.00 73.02 C ATOM 2197 C GLY A 1077 −18.543 69.995 28.392 1.00 72.32 C ATOM 2198 O GLY A 1077 −18.322 70.690 29.387 1.00 73.12 O ATOM 2199 N ILE A 1078 −18.523 68.661 28.399 1.00 72.41 N ATOM 2200 CA ILE A 1078 −18.104 67.858 29.564 1.00 73.34 C ATOM 2201 C ILE A 1078 −16.739 68.293 30.083 1.00 71.54 C ATOM 2202 O ILE A 1078 −16.577 68.522 31.280 1.00 76.16 O ATOM 2203 CB ILE A 1078 −18.044 66.363 29.210 1.00 74.38 C ATOM 2204 CG1 ILE A 1078 −19.453 65.757 29.198 1.00 79.15 C ATOM 2205 CG2 ILE A 1078 −17.205 65.616 30.174 1.00 64.66 C ATOM 2206 CD1 ILE A 1078 −19.514 64.385 28.554 1.00 75.65 C ATOM 2207 N LEU A 1079 −15.775 68.407 29.177 1.00 72.22 N ATOM 2208 CA LEU A 1079 −14.407 68.819 29.512 1.00 73.11 C ATOM 2209 C LEU A 1079 −14.278 70.275 30.001 1.00 73.92 C ATOM 2210 O LEU A 1079 −13.281 70.613 30.627 1.00 78.75 O ATOM 2211 CB LEU A 1079 −13.462 68.578 28.317 1.00 72.14 C ATOM 2212 CG LEU A 1079 −13.192 67.115 27.909 1.00 72.35 C ATOM 2213 CD1 LEU A 1079 −12.301 67.074 26.679 1.00 59.40 C ATOM 2214 CD2 LEU A 1079 −12.543 66.309 29.031 1.00 62.86 C ATOM 2215 N ARG A 1080 −15.269 71.118 29.714 1.00 73.61 N ATOM 2216 CA ARG A 1080 −15.330 72.490 30.251 1.00 74.19 C ATOM 2217 C ARG A 1080 −16.039 72.537 31.609 1.00 72.82 C ATOM 2218 O ARG A 1080 −15.894 73.505 32.331 1.00 72.73 O ATOM 2219 CB ARG A 1080 −16.061 73.439 29.301 1.00 74.26 C ATOM 2220 CG ARG A 1080 −15.236 73.955 28.140 1.00 79.49 C ATOM 2221 CD ARG A 1080 −15.964 75.121 27.460 1.00 79.38 C ATOM 2222 NE ARG A 1080 −17.260 74.691 26.922 1.00 88.39 N ATOM 2223 CZ ARG A 1080 −17.535 74.387 25.636 1.00 87.86 C ATOM 2224 NH1 ARG A 1080 −16.608 74.451 24.665 1.00 87.22 N ATOM 2225 NH2 ARG A 1080 −18.777 74.009 25.314 1.00 81.94 N ATOM 2226 N ASN A 1081 −16.800 71.495 31.946 1.00 72.20 N ATOM 2227 CA ASN A 1081 −17.553 71.464 33.183 1.00 70.14 C ATOM 2228 C ASN A 1081 −16.680 70.903 34.293 1.00 70.99 C ATOM 2229 O ASN A 1081 −16.295 69.733 34.252 1.00 70.87 O ATOM 2230 CB ASN A 1081 −18.817 70.642 33.002 1.00 68.83 C ATOM 2231 CG ASN A 1081 −19.789 70.813 34.137 1.00 74.52 C ATOM 2232 OD1 ASN A 1081 −19.521 70.405 35.275 1.00 81.88 O ATOM 2233 ND2 ASN A 1081 −20.937 71.417 33.848 1.00 65.69 N ATOM 2234 N ALA A 1082 −16.374 71.750 35.281 1.00 69.81 N ATOM 2235 CA ALA A 1082 −15.539 71.386 36.459 1.00 71.34 C ATOM 2236 C ALA A 1082 −15.942 70.101 37.203 1.00 70.68 C ATOM 2237 O ALA A 1082 −15.081 69.426 37.748 1.00 77.40 O ATOM 2238 CB ALA A 1082 −15.508 72.546 37.451 1.00 64.36 C ATOM 2239 N LYS A 1083 −17.233 69.784 37.213 1.00 68.55 N ATOM 2240 CA LYS A 1083 −17.776 68.623 37.931 1.00 69.05 C ATOM 2241 C LYS A 1083 −17.798 67.337 37.093 1.00 72.30 C ATOM 2242 O LYS A 1083 −17.732 66.243 37.649 1.00 76.42 O ATOM 2243 CB LYS A 1083 −19.193 68.923 38.398 1.00 70.45 C ATOM 2244 CG LYS A 1083 −19.359 70.297 39.063 1.00 75.92 C ATOM 2245 CD LYS A 1083 −20.720 70.447 39.698 1.00 81.44 C ATOM 2246 CE LYS A 1083 −20.926 71.864 40.217 1.00 85.50 C ATOM 2247 NZ LYS A 1083 −22.148 71.994 41.068 1.00 88.29 N ATOM 2248 N LEU A 1084 −17.891 67.460 35.765 1.00 68.40 N ATOM 2249 CA LEU A 1084 −17.898 66.286 34.894 1.00 70.90 C ATOM 2250 C LEU A 1084 −16.518 65.924 34.402 1.00 70.17 C ATOM 2251 O LEU A 1084 −16.247 64.740 34.205 1.00 72.79 O ATOM 2252 CB LEU A 1084 −18.843 66.497 33.694 1.00 68.64 C ATOM 2253 CG LEU A 1084 −20.304 66.801 34.051 1.00 71.12 C ATOM 2254 CD1 LEU A 1084 −21.213 66.916 32.787 1.00 59.19 C ATOM 2255 CD2 LEU A 1084 −20.847 65.747 35.010 1.00 56.68 C ATOM 2256 N LYS A 1085 −15.650 66.917 34.205 1.00 68.34 N ATOM 2257 CA LYS A 1085 −14.316 66.663 33.652 1.00 68.63 C ATOM 2258 C LYS A 1085 −13.538 65.559 34.383 1.00 68.76 C ATOM 2259 O LYS A 1085 −13.070 64.643 33.723 1.00 75.06 O ATOM 2260 CB LYS A 1085 −13.482 67.951 33.565 1.00 67.89 C ATOM 2261 CG LYS A 1085 −12.110 67.770 32.917 1.00 69.24 C ATOM 2262 CD LYS A 1085 −11.322 69.054 32.925 1.00 72.42 C ATOM 2263 CE LYS A 1085 −10.040 68.905 32.138 1.00 77.84 C ATOM 2264 NZ LYS A 1085 −9.131 67.881 32.745 1.00 85.14 N ATOM 2265 N PRO A 1086 −13.399 65.636 35.740 1.00 69.24 N ATOM 2266 CA PRO A 1086 −12.637 64.586 36.440 1.00 69.18 C ATOM 2267 C PRO A 1086 −13.226 63.190 36.261 1.00 68.97 C ATOM 2268 O PRO A 1086 −12.488 62.197 36.199 1.00 69.99 O ATOM 2269 CB PRO A 1086 −12.771 64.966 37.923 1.00 67.72 C ATOM 2270 CG PRO A 1086 −13.201 66.357 37.950 1.00 72.53 C ATOM 2271 CD PRO A 1086 −13.918 66.646 36.679 1.00 66.17 C ATOM 2272 N VAL A 1087 −14.549 63.136 36.184 1.00 68.84 N ATOM 2273 CA VAL A 1087 −15.265 61.885 36.092 1.00 72.28 C ATOM 2274 C VAL A 1087 −14.985 61.291 34.743 1.00 73.91 C ATOM 2275 O VAL A 1087 −14.480 60.176 34.672 1.00 77.11 O ATOM 2276 CB VAL A 1087 −16.769 62.062 36.305 1.00 72.36 C ATOM 2277 CG1 VAL A 1087 −17.431 60.720 36.368 1.00 78.50 C ATOM 2278 CG2 VAL A 1087 −17.032 62.832 37.602 1.00 75.48 C ATOM 2279 N TYR A 1088 −15.310 62.047 33.691 1.00 76.42 N ATOM 2280 CA TYR A 1088 −15.087 61.660 32.290 1.00 74.22 C ATOM 2281 C TYR A 1088 −13.655 61.241 32.054 1.00 73.79 C ATOM 2282 O TYR A 1088 −13.414 60.184 31.502 1.00 71.26 O ATOM 2283 CB TYR A 1088 −15.420 62.840 31.382 1.00 76.80 C ATOM 2284 CG TYR A 1088 −15.345 62.536 29.909 1.00 78.30 C ATOM 2285 CD1 TYR A 1088 −16.447 61.994 29.225 1.00 82.88 C ATOM 2286 CD2 TYR A 1088 −14.181 62.787 29.185 1.00 82.17 C ATOM 2287 CE1 TYR A 1088 −16.368 61.712 27.838 1.00 78.90 C ATOM 2288 CE2 TYR A 1088 −14.102 62.511 27.823 1.00 73.71 C ATOM 2289 CZ TYR A 1088 −15.199 61.976 27.162 1.00 71.96 C ATOM 2290 OH TYR A 1088 −15.099 61.705 25.804 1.00 90.19 O ATOM 2291 N ASP A 1089 −12.708 62.082 32.483 1.00 73.28 N ATOM 2292 CA ASP A 1089 −11.262 61.780 32.399 1.00 72.57 C ATOM 2293 C ASP A 1089 −10.880 60.403 32.936 1.00 71.20 C ATOM 2294 O ASP A 1089 −9.987 59.755 32.407 1.00 70.55 O ATOM 2295 CB ASP A 1089 −10.438 62.802 33.208 1.00 72.85 C ATOM 2296 CG ASP A 1089 −10.162 64.118 32.468 1.00 79.93 C ATOM 2297 OD1 ASP A 1089 −10.551 64.287 31.274 1.00 78.98 O ATOM 2298 OD2 ASP A 1089 −9.528 65.004 33.120 1.00 69.59 O ATOM 2299 N SER A 1090 −11.562 59.969 33.993 1.00 72.25 N ATOM 2300 CA SER A 1090 −11.244 58.711 34.670 1.00 71.13 C ATOM 2301 C SER A 1090 −11.893 57.495 34.027 1.00 69.01 C ATOM 2302 O SER A 1090 −11.411 56.383 34.199 1.00 69.79 O ATOM 2303 CB SER A 1090 −11.684 58.791 36.124 1.00 67.58 C ATOM 2304 OG SER A 1090 −13.085 58.665 36.232 1.00 65.84 O ATOM 2305 N LEU A 1091 −12.979 57.703 33.293 1.00 72.26 N ATOM 2306 CA LEU A 1091 −13.727 56.593 32.729 1.00 70.30 C ATOM 2307 C LEU A 1091 −13.092 56.023 31.462 1.00 73.59 C ATOM 2308 O LEU A 1091 −12.420 56.715 30.705 1.00 75.14 O ATOM 2309 CB LEU A 1091 −15.172 57.014 32.457 1.00 73.21 C ATOM 2310 CG LEU A 1091 −16.060 57.298 33.687 1.00 69.70 C ATOM 2311 CD1 LEU A 1091 −17.374 57.869 33.211 1.00 74.59 C ATOM 2312 CD2 LEU A 1091 −16.287 56.051 34.482 1.00 61.23 C ATOM 2313 N ASP A 1092 −13.324 54.735 31.257 1.00 73.36 N ATOM 2314 CA ASP A 1092 −12.955 54.046 30.017 1.00 74.54 C ATOM 2315 C ASP A 1092 −13.910 54.480 28.927 1.00 72.07 C ATOM 2316 O ASP A 1092 −14.938 55.088 29.230 1.00 69.09 O ATOM 2317 CB ASP A 1092 −13.093 52.547 30.180 1.00 74.48 C ATOM 2318 CG ASP A 1092 −14.502 52.157 30.522 1.00 82.40 C ATOM 2319 OD1 ASP A 1092 −15.393 52.059 29.643 1.00 78.10 O ATOM 2320 OD2 ASP A 1092 −14.702 51.956 31.702 1.00 93.39 O ATOM 2321 N ALA A 1093 −13.561 54.156 27.677 1.00 71.26 N ATOM 2322 CA ALA A 1093 −14.332 54.530 26.455 1.00 72.91 C ATOM 2323 C ALA A 1093 −15.817 54.185 26.482 1.00 72.49 C ATOM 2324 O ALA A 1093 −16.642 55.016 26.143 1.00 77.77 O ATOM 2325 CB ALA A 1093 −13.682 53.884 25.213 1.00 68.92 C ATOM 2326 N VAL A 1094 −16.152 52.959 26.882 1.00 74.25 N ATOM 2327 CA VAL A 1094 −17.566 52.517 26.970 1.00 76.09 C ATOM 2328 C VAL A 1094 −18.388 53.297 28.021 1.00 74.99 C ATOM 2329 O VAL A 1094 −19.550 53.686 27.760 1.00 74.98 O ATOM 2330 CB VAL A 1094 −17.680 51.014 27.268 1.00 77.56 C ATOM 2331 CG1 VAL A 1094 −19.124 50.634 27.518 1.00 80.02 C ATOM 2332 CG2 VAL A 1094 −17.091 50.192 26.098 1.00 77.82 C ATOM 2333 N ARG A 1095 −17.804 53.527 29.192 1.00 74.55 N ATOM 2334 CA ARG A 1095 −18.502 54.306 30.245 1.00 72.87 C ATOM 2335 C ARG A 1095 −18.564 55.792 29.877 1.00 70.86 C ATOM 2336 O ARG A 1095 −19.529 56.471 30.224 1.00 70.60 O ATOM 2337 CB ARG A 1095 −17.855 54.090 31.598 1.00 74.90 C ATOM 2338 CG ARG A 1095 −17.944 52.648 32.049 1.00 74.88 C ATOM 2339 CD ARG A 1095 −17.456 52.519 33.465 1.00 76.13 C ATOM 2340 NE ARG A 1095 −17.765 51.222 34.039 1.00 68.67 N ATOM 2341 CZ ARG A 1095 −17.035 50.111 33.935 1.00 75.88 C ATOM 2342 NH1 ARG A 1095 −15.900 50.077 33.261 1.00 77.10 N ATOM 2343 NH2 ARG A 1095 −17.456 48.999 34.523 1.00 78.82 N ATOM 2344 N ARG A 1096 −17.550 56.304 29.176 1.00 70.40 N ATOM 2345 CA ARG A 1096 −17.646 57.654 28.616 1.00 72.78 C ATOM 2346 C ARG A 1096 −18.895 57.781 27.683 1.00 75.95 C ATOM 2347 O ARG A 1096 −19.603 58.790 27.741 1.00 76.20 O ATOM 2348 CB ARG A 1096 −16.382 58.032 27.849 1.00 74.96 C ATOM 2349 CG ARG A 1096 −15.212 58.381 28.729 1.00 76.80 C ATOM 2350 CD ARG A 1096 −13.987 58.699 27.888 1.00 73.69 C ATOM 2351 NE ARG A 1096 −12.821 58.877 28.724 1.00 75.05 N ATOM 2352 CZ ARG A 1096 −11.617 59.276 28.334 1.00 76.09 C ATOM 2353 NH1 ARG A 1096 −11.358 59.568 27.062 1.00 79.66 N ATOM 2354 NH2 ARG A 1096 −10.642 59.388 29.244 1.00 73.76 N ATOM 2355 N ALA A 1097 −19.154 56.767 26.842 1.00 74.52 N ATOM 2356 CA ALA A 1097 −20.381 56.762 25.993 1.00 75.40 C ATOM 2357 C ALA A 1097 −21.606 56.787 26.899 1.00 74.68 C ATOM 2358 O ALA A 1097 −22.596 57.444 26.594 1.00 75.19 O ATOM 2359 CB ALA A 1097 −20.428 55.556 25.084 1.00 65.57 C ATOM 2360 N ALA A 1098 −21.538 56.068 28.019 1.00 77.29 N ATOM 2361 CA ALA A 1098 −22.627 56.110 29.004 1.00 77.63 C ATOM 2362 C ALA A 1098 −22.855 57.549 29.425 1.00 75.46 C ATOM 2363 O ALA A 1098 −23.953 58.037 29.306 1.00 77.90 O ATOM 2364 CB ALA A 1098 −22.319 55.252 30.201 1.00 72.80 C ATOM 2365 N LEU A 1099 −21.808 58.215 29.906 1.00 74.27 N ATOM 2366 CA LEU A 1099 −21.935 59.610 30.389 1.00 73.67 C ATOM 2367 C LEU A 1099 −22.493 60.542 29.329 1.00 73.73 C ATOM 2368 O LEU A 1099 −23.412 61.328 29.602 1.00 73.92 O ATOM 2369 CB LEU A 1099 −20.590 60.144 30.886 1.00 76.16 C ATOM 2370 CG LEU A 1099 −20.612 61.381 31.794 1.00 76.75 C ATOM 2371 CD1 LEU A 1099 −21.495 61.167 33.000 1.00 72.22 C ATOM 2372 CD2 LEU A 1099 −19.187 61.741 32.236 1.00 66.69 C ATOM 2373 N ILE A 1100 −21.941 60.454 28.115 1.00 72.03 N ATOM 2374 CA ILE A 1100 −22.430 61.252 26.986 1.00 70.39 C ATOM 2375 C ILE A 1100 −23.908 60.998 26.758 1.00 69.34 C ATOM 2376 O ILE A 1100 −24.674 61.941 26.552 1.00 70.67 O ATOM 2377 CB ILE A 1100 −21.672 60.964 25.677 1.00 74.51 C ATOM 2378 CG1 ILE A 1100 −20.208 61.411 25.789 1.00 74.12 C ATOM 2379 CG2 ILE A 1100 −22.368 61.684 24.523 1.00 66.55 C ATOM 2380 CD1 ILE A 1100 −19.273 60.643 24.910 1.00 71.96 C ATOM 2381 N ASN A 1101 −24.305 59.727 26.798 1.00 69.64 N ATOM 2382 CA ASN A 1101 −25.716 59.340 26.656 1.00 71.15 C ATOM 2383 C ASN A 1101 −26.587 60.154 27.596 1.00 73.26 C ATOM 2384 O ASN A 1101 −27.606 60.720 27.158 1.00 73.01 O ATOM 2385 CB ASN A 1101 −25.859 57.819 26.880 1.00 74.91 C ATOM 2386 CG ASN A 1101 −27.242 57.286 26.621 1.00 72.41 C ATOM 2387 OD1 ASN A 1101 −28.218 57.895 26.974 1.00 68.21 O ATOM 2388 ND2 ASN A 1101 −27.321 56.110 25.995 1.00 66.91 N ATOM 2389 N MET A 1102 −26.191 60.217 28.876 1.00 73.18 N ATOM 2390 CA MET A 1102 −26.922 60.986 29.894 1.00 73.53 C ATOM 2391 C MET A 1102 −26.897 62.472 29.579 1.00 72.72 C ATOM 2392 O MET A 1102 −27.909 63.130 29.719 1.00 75.34 O ATOM 2393 CB MET A 1102 −26.341 60.788 31.315 1.00 69.92 C ATOM 2394 CG MET A 1102 −26.311 59.343 31.846 1.00 71.61 C ATOM 2395 SD MET A 1102 −25.639 59.205 33.558 1.00 75.01 S ATOM 2396 CE MET A 1102 −26.903 60.133 34.436 1.00 72.22 C ATOM 2397 N VAL A 1103 −25.743 62.997 29.160 1.00 73.08 N ATOM 2398 CA VAL A 1103 −25.650 64.430 28.774 1.00 75.10 C ATOM 2399 C VAL A 1103 −26.605 64.736 27.585 1.00 74.50 C ATOM 2400 O VAL A 1103 −27.280 65.769 27.587 1.00 74.33 O ATOM 2401 CB VAL A 1103 −24.203 64.859 28.445 1.00 74.63 C ATOM 2402 CG1 VAL A 1103 −24.181 66.214 27.773 1.00 74.66 C ATOM 2403 CG2 VAL A 1103 −23.345 64.892 29.715 1.00 73.28 C ATOM 2404 N PHE A 1104 −26.648 63.838 26.598 1.00 72.50 N ATOM 2405 CA PHE A 1104 −27.641 63.937 25.507 1.00 77.14 C ATOM 2406 C PHE A 1104 −29.050 64.133 26.087 1.00 77.59 C ATOM 2407 O PHE A 1104 −29.746 65.023 25.684 1.00 78.04 O ATOM 2408 CB PHE A 1104 −27.615 62.712 24.557 1.00 78.31 C ATOM 2409 CG PHE A 1104 −26.870 62.933 23.259 1.00 81.99 C ATOM 2410 CD1 PHE A 1104 −25.489 63.029 23.238 1.00 78.79 C ATOM 2411 CD2 PHE A 1104 −27.580 63.037 22.027 1.00 90.20 C ATOM 2412 CE1 PHE A 1104 −24.800 63.227 22.021 1.00 77.16 C ATOM 2413 CE2 PHE A 1104 −26.910 63.237 20.821 1.00 84.00 C ATOM 2414 CZ PHE A 1104 −25.509 63.332 20.823 1.00 86.09 C ATOM 2415 N GLN A 1105 −29.446 63.296 27.039 1.00 76.01 N ATOM 2416 CA GLN A 1105 −30.818 63.335 27.575 1.00 75.44 C ATOM 2417 C GLN A 1105 −31.159 64.480 28.544 1.00 74.63 C ATOM 2418 O GLN A 1105 −32.249 65.043 28.455 1.00 75.26 O ATOM 2419 CB GLN A 1105 −31.100 62.012 28.270 1.00 77.85 C ATOM 2420 CG GLN A 1105 −32.567 61.757 28.615 1.00 75.54 C ATOM 2421 CD GLN A 1105 −32.781 60.378 29.168 1.00 75.71 C ATOM 2422 OE1 GLN A 1105 −31.834 59.620 29.340 1.00 71.29 O ATOM 2423 NE2 GLN A 1105 −34.025 60.039 29.450 1.00 70.96 N ATOM 2424 N MET A 1106 −30.250 64.819 29.456 1.00 76.62 N ATOM 2425 CA MET A 1106 −30.529 65.838 30.524 1.00 77.20 C ATOM 2426 C MET A 1106 −29.666 67.100 30.498 1.00 75.43 C ATOM 2427 O MET A 1106 −29.901 68.010 31.296 1.00 78.05 O ATOM 2428 CB MET A 1106 −30.319 65.216 31.902 1.00 79.39 C ATOM 2429 CG MET A 1106 −30.886 63.832 32.124 1.00 85.85 C ATOM 2430 SD MET A 1106 −30.091 63.064 33.542 1.00 85.72 S ATOM 2431 CE MET A 1106 −29.933 61.419 32.917 1.00 81.10 C ATOM 2432 N GLY A 1107 −28.675 67.166 29.609 1.00 75.52 N ATOM 2433 CA GLY A 1107 −27.748 68.294 29.560 1.00 76.26 C ATOM 2434 C GLY A 1107 −26.702 68.166 30.651 1.00 78.60 C ATOM 2435 O GLY A 1107 −26.827 67.330 31.555 1.00 78.11 O ATOM 2436 N GLU A 1108 −25.666 68.993 30.573 1.00 79.86 N ATOM 2437 CA GLU A 1108 −24.556 68.931 31.548 1.00 82.66 C ATOM 2438 C GLU A 1108 −24.966 69.224 32.966 1.00 82.97 C ATOM 2439 O GLU A 1108 −24.495 68.560 33.891 1.00 86.76 O ATOM 2440 CB GLU A 1108 −23.454 69.901 31.185 1.00 82.78 C ATOM 2441 CG GLU A 1108 −22.714 69.512 29.969 1.00 86.49 C ATOM 2442 CD GLU A 1108 −21.722 70.556 29.554 1.00 90.68 C ATOM 2443 OE1 GLU A 1108 −21.148 71.271 30.409 1.00 90.93 O ATOM 2444 OE2 GLU A 1108 −21.523 70.659 28.348 1.00 103.73 O ATOM 2445 N THR A 1109 −25.837 70.214 33.132 1.00 82.33 N ATOM 2446 CA THR A 1109 −26.324 70.597 34.451 1.00 80.83 C ATOM 2447 C THR A 1109 −27.061 69.455 35.112 1.00 79.03 C ATOM 2448 O THR A 1109 −26.755 69.098 36.247 1.00 84.70 O ATOM 2449 CB THR A 1109 −27.270 71.802 34.386 1.00 79.62 C ATOM 2450 OG1 THR A 1109 −26.591 72.907 33.776 1.00 80.39 O ATOM 2451 CG2 THR A 1109 −27.731 72.196 35.790 1.00 81.01 C ATOM 2452 N GLY A 1110 −28.028 68.889 34.395 1.00 78.14 N ATOM 2453 CA GLY A 1110 −28.794 67.749 34.888 1.00 77.59 C ATOM 2454 C GLY A 1110 −27.893 66.637 35.399 1.00 76.91 C ATOM 2455 O GLY A 1110 −28.147 66.069 36.450 1.00 77.84 O ATOM 2456 N VAL A 1111 −26.838 66.348 34.639 1.00 74.81 N ATOM 2457 CA VAL A 1111 −25.901 65.274 34.952 1.00 73.76 C ATOM 2458 C VAL A 1111 −24.901 65.663 36.063 1.00 74.74 C ATOM 2459 O VAL A 1111 −24.475 64.809 36.863 1.00 70.56 O ATOM 2460 CB VAL A 1111 −25.094 64.844 33.715 1.00 68.49 C ATOM 2461 CG1 VAL A 1111 −24.173 63.695 34.081 1.00 64.99 C ATOM 2462 CG2 VAL A 1111 −26.023 64.438 32.582 1.00 68.09 C ATOM 2463 N ALA A 1112 −24.531 66.949 36.096 1.00 74.29 N ATOM 2464 CA ALA A 1112 −23.631 67.491 37.119 1.00 77.63 C ATOM 2465 C ALA A 1112 −24.255 67.423 38.496 1.00 77.53 C ATOM 2466 O ALA A 1112 −23.542 67.410 39.483 1.00 82.24 O ATOM 2467 CB ALA A 1112 −23.256 68.931 36.798 1.00 77.58 C ATOM 2468 N GLY A 1113 −25.591 67.381 38.553 1.00 79.76 N ATOM 2469 CA GLY A 1113 −26.323 67.270 39.808 1.00 77.64 C ATOM 2470 C GLY A 1113 −26.267 65.900 40.461 1.00 78.25 C ATOM 2471 O GLY A 1113 −26.687 65.780 41.588 1.00 76.50 O ATOM 2472 N PHE A 1114 −25.757 64.861 39.774 1.00 77.88 N ATOM 2473 CA PHE A 1114 −25.586 63.533 40.383 1.00 76.03 C ATOM 2474 C PHE A 1114 −24.276 63.500 41.179 1.00 75.36 C ATOM 2475 O PHE A 1114 −23.399 62.693 40.887 1.00 73.89 O ATOM 2476 CB PHE A 1114 −25.559 62.437 39.310 1.00 81.97 C ATOM 2477 CG PHE A 1114 −26.845 62.247 38.594 1.00 84.39 C ATOM 2478 CD1 PHE A 1114 −27.906 61.609 39.202 1.00 93.33 C ATOM 2479 CD2 PHE A 1114 −27.001 62.696 37.310 1.00 90.99 C ATOM 2480 CE1 PHE A 1114 −29.108 61.433 38.521 1.00 91.37 C ATOM 2481 CE2 PHE A 1114 −28.187 62.526 36.627 1.00 91.29 C ATOM 2482 CZ PHE A 1114 −29.244 61.894 37.229 1.00 89.69 C ATOM 2483 N THR A 1115 −24.139 64.364 42.186 1.00 70.77 N ATOM 2484 CA THR A 1115 −22.860 64.550 42.845 1.00 71.53 C ATOM 2485 C THR A 1115 −22.292 63.256 43.420 1.00 72.52 C ATOM 2486 O THR A 1115 −21.124 62.964 43.206 1.00 74.92 O ATOM 2487 CB THR A 1115 −22.950 65.613 43.957 1.00 74.18 C ATOM 2488 OG1 THR A 1115 −23.638 66.761 43.454 1.00 81.29 O ATOM 2489 CG2 THR A 1115 −21.562 66.027 44.419 1.00 65.46 C ATOM 2490 N ASN A 1116 −23.112 62.494 44.138 1.00 73.53 N ATOM 2491 CA ASN A 1116 −22.644 61.260 44.779 1.00 74.43 C ATOM 2492 C ASN A 1116 −22.327 60.176 43.753 1.00 74.36 C ATOM 2493 O ASN A 1116 −21.286 59.523 43.854 1.00 73.98 O ATOM 2494 CB ASN A 1116 −23.652 60.723 45.803 1.00 76.08 C ATOM 2495 CG ASN A 1116 −23.916 61.701 46.958 1.00 81.08 C ATOM 2496 OD1 ASN A 1116 −22.994 62.125 47.647 1.00 76.24 O ATOM 2497 ND2 ASN A 1116 −25.181 62.050 47.163 1.00 82.05 N ATOM 2498 N SER A 1117 −23.218 59.986 42.776 1.00 71.66 N ATOM 2499 CA SER A 1117 −22.990 59.014 41.697 1.00 71.35 C ATOM 2500 C SER A 1117 −21.736 59.307 40.895 1.00 72.60 C ATOM 2501 O SER A 1117 −21.010 58.386 40.567 1.00 73.79 O ATOM 2502 CB SER A 1117 −24.170 58.962 40.748 1.00 74.52 C ATOM 2503 OG SER A 1117 −25.271 58.329 41.351 1.00 74.10 O ATOM 2504 N LEU A 1118 −21.484 60.580 40.586 1.00 74.22 N ATOM 2505 CA LEU A 1118 −20.253 60.997 39.863 1.00 74.12 C ATOM 2506 C LEU A 1118 −18.961 60.660 40.618 1.00 73.96 C ATOM 2507 O LEU A 1118 −17.984 60.195 40.025 1.00 78.17 O ATOM 2508 CB LEU A 1118 −20.299 62.503 39.566 1.00 74.92 C ATOM 2509 CG LEU A 1118 −21.337 62.937 38.500 1.00 77.06 C ATOM 2510 CD1 LEU A 1118 −21.516 64.484 38.518 1.00 79.91 C ATOM 2511 CD2 LEU A 1118 −20.950 62.456 37.120 1.00 79.15 C ATOM 2512 N ARG A 1119 −18.972 60.901 41.917 1.00 73.81 N ATOM 2513 CA ARG A 1119 −17.857 60.571 42.806 1.00 76.37 C ATOM 2514 C ARG A 1119 −17.586 59.069 42.837 1.00 73.72 C ATOM 2515 O ARG A 1119 −16.439 58.633 42.760 1.00 71.54 O ATOM 2516 CB ARG A 1119 −18.179 61.041 44.229 1.00 72.72 C ATOM 2517 CG ARG A 1119 −17.067 60.873 45.241 1.00 81.26 C ATOM 2518 CD ARG A 1119 −17.608 60.978 46.712 1.00 88.03 C ATOM 2519 NE ARG A 1119 −18.622 62.027 46.876 1.00 97.80 N ATOM 2520 CZ ARG A 1119 −18.387 63.350 46.852 1.00 107.35 C ATOM 2521 NH1 ARG A 1119 −17.149 63.849 46.666 1.00 110.91 N ATOM 2522 NH2 ARG A 1119 −19.407 64.207 47.016 1.00 108.55 N ATOM 2523 N MET A 1120 −18.653 58.296 42.958 1.00 72.40 N ATOM 2524 CA MET A 1120 −18.554 56.843 42.948 1.00 75.44 C ATOM 2525 C MET A 1120 −18.059 56.323 41.610 1.00 72.59 C ATOM 2526 O MET A 1120 −17.199 55.455 41.580 1.00 74.45 O ATOM 2527 CB MET A 1120 −19.884 56.228 43.338 1.00 74.73 C ATOM 2528 CG MET A 1120 −20.116 56.407 44.861 1.00 79.31 C ATOM 2529 SD MET A 1120 −21.698 55.810 45.341 1.00 86.73 S ATOM 2530 CE MET A 1120 −22.843 56.943 44.571 1.00 82.05 C ATOM 2531 N LEU A 1121 −18.592 56.851 40.516 1.00 70.51 N ATOM 2532 CA LEU A 1121 −18.067 56.495 39.211 1.00 73.43 C ATOM 2533 C LEU A 1121 −16.581 56.816 39.149 1.00 71.65 C ATOM 2534 O LEU A 1121 −15.810 55.970 38.728 1.00 74.87 O ATOM 2535 CB LEU A 1121 −18.802 57.213 38.086 1.00 67.49 C ATOM 2536 CG LEU A 1121 −20.247 56.734 37.877 1.00 72.80 C ATOM 2537 CD1 LEU A 1121 −20.990 57.724 36.968 1.00 65.17 C ATOM 2538 CD2 LEU A 1121 −20.343 55.241 37.364 1.00 62.04 C ATOM 2539 N GLN A 1122 −16.186 58.020 39.565 1.00 69.51 N ATOM 2540 CA GLN A 1122 −14.760 58.419 39.551 1.00 69.43 C ATOM 2541 C GLN A 1122 −13.852 57.422 40.302 1.00 69.96 C ATOM 2542 O GLN A 1122 −12.726 57.188 39.889 1.00 72.67 O ATOM 2543 CB GLN A 1122 −14.602 59.828 40.123 1.00 69.75 C ATOM 2544 CG GLN A 1122 −13.203 60.432 39.975 1.00 73.87 C ATOM 2545 CD GLN A 1122 −13.125 61.883 40.443 1.00 79.31 C ATOM 2546 OE1 GLN A 1122 −14.139 62.505 40.758 1.00 88.60 O ATOM 2547 NE2 GLN A 1122 −11.907 62.426 40.490 1.00 84.85 N ATOM 2548 N GLN A 1123 −14.364 56.850 41.395 1.00 66.33 N ATOM 2549 CA GLN A 1123 −13.659 55.833 42.182 1.00 71.40 C ATOM 2550 C GLN A 1123 −13.857 54.373 41.688 1.00 70.37 C ATOM 2551 O GLN A 1123 −13.323 53.450 42.300 1.00 68.04 O ATOM 2552 CB GLN A 1123 −14.083 55.958 43.662 1.00 66.15 C ATOM 2553 CG GLN A 1123 −13.701 57.318 44.267 1.00 65.21 C ATOM 2554 CD GLN A 1123 −14.267 57.566 45.652 1.00 69.71 C ATOM 2555 OE1 GLN A 1123 −15.294 57.018 46.035 1.00 77.32 O ATOM 2556 NE2 GLN A 1123 −13.585 58.404 46.411 1.00 63.29 N ATOM 2557 N LYS A 1124 −14.609 54.174 40.604 1.00 69.50 N ATOM 2558 CA LYS A 1124 −14.896 52.830 40.066 1.00 74.09 C ATOM 2559 C LYS A 1124 −15.719 51.959 41.048 1.00 75.70 C ATOM 2560 O LYS A 1124 −15.562 50.733 41.084 1.00 75.06 O ATOM 2561 CB LYS A 1124 −13.603 52.089 39.688 1.00 79.02 C ATOM 2562 CG LYS A 1124 −12.571 52.892 38.892 1.00 83.65 C ATOM 2563 CD LYS A 1124 −12.682 52.701 37.399 1.00 89.63 C ATOM 2564 CE LYS A 1124 −11.978 53.758 36.540 1.00 92.96 C ATOM 2565 NZ LYS A 1124 −12.833 55.011 36.416 1.00 104.91 N ATOM 2566 N ARG A 1125 −16.586 52.609 41.828 1.00 74.41 N ATOM 2567 CA ARG A 1125 −17.489 51.958 42.758 1.00 73.39 C ATOM 2568 C ARG A 1125 −18.787 51.862 41.975 1.00 72.92 C ATOM 2569 O ARG A 1125 −19.755 52.569 42.238 1.00 75.78 O ATOM 2570 CB ARG A 1125 −17.625 52.765 44.049 1.00 72.14 C ATOM 2571 CG ARG A 1125 −16.304 52.959 44.776 1.00 74.10 C ATOM 2572 CD ARG A 1125 −16.448 53.860 45.974 1.00 76.04 C ATOM 2573 NE ARG A 1125 −17.144 53.226 47.096 1.00 83.94 N ATOM 2574 CZ ARG A 1125 −17.567 53.859 48.203 1.00 87.62 C ATOM 2575 NH1 ARG A 1125 −17.373 55.179 48.365 1.00 91.97 N ATOM 2576 NH2 ARG A 1125 −18.195 53.171 49.172 1.00 84.34 N ATOM 2577 N TRP A 1126 −18.778 50.963 41.005 1.00 74.20 N ATOM 2578 CA TRP A 1126 −19.839 50.847 39.981 1.00 72.97 C ATOM 2579 C TRP A 1126 −21.220 50.507 40.541 1.00 71.90 C ATOM 2580 O TRP A 1126 −22.203 51.187 40.232 1.00 67.72 O ATOM 2581 CB TRP A 1126 −19.473 49.781 38.959 1.00 69.76 C ATOM 2582 CG TRP A 1126 −18.124 49.894 38.335 1.00 70.87 C ATOM 2583 CD1 TRP A 1126 −17.183 48.917 38.255 1.00 66.79 C ATOM 2584 CD2 TRP A 1126 −17.564 51.047 37.699 1.00 70.96 C ATOM 2585 NE1 TRP A 1126 −16.068 49.386 37.610 1.00 69.94 N ATOM 2586 CE2 TRP A 1126 −16.280 50.692 37.260 1.00 70.31 C ATOM 2587 CE3 TRP A 1126 −18.026 52.351 37.457 1.00 71.68 C ATOM 2588 CZ2 TRP A 1126 −15.460 51.576 36.600 1.00 74.24 C ATOM 2589 CZ3 TRP A 1126 −17.205 53.235 36.801 1.00 71.20 C ATOM 2590 CH2 TRP A 1126 −15.939 52.850 36.379 1.00 73.75 C ATOM 2591 N ASP A 1127 −21.273 49.458 41.357 1.00 72.11 N ATOM 2592 CA ASP A 1127 −22.537 49.012 41.994 1.00 74.28 C ATOM 2593 C ASP A 1127 −23.168 50.100 42.830 1.00 72.96 C ATOM 2594 O ASP A 1127 −24.377 50.283 42.781 1.00 77.35 O ATOM 2595 CB ASP A 1127 −22.326 47.768 42.874 1.00 73.85 C ATOM 2596 CG ASP A 1127 −22.177 46.475 42.065 1.00 80.36 C ATOM 2597 OD1 ASP A 1127 −22.034 46.530 40.836 1.00 81.11 O ATOM 2598 OD2 ASP A 1127 −22.204 45.386 42.681 1.00 97.24 O ATOM 2599 N GLU A 1128 −22.349 50.813 43.586 1.00 70.74 N ATOM 2600 CA GLU A 1128 −22.840 51.886 44.452 1.00 72.33 C ATOM 2601 C GLU A 1128 −23.299 53.089 43.646 1.00 70.63 C ATOM 2602 O GLU A 1128 −24.276 53.740 44.017 1.00 70.95 O ATOM 2603 CB GLU A 1128 −21.764 52.293 45.420 1.00 70.41 C ATOM 2604 CG GLU A 1128 −21.384 51.186 46.364 1.00 79.81 C ATOM 2605 CD GLU A 1128 −20.232 51.570 47.219 1.00 81.59 C ATOM 2606 OE1 GLU A 1128 −20.445 52.476 48.059 1.00 90.24 O ATOM 2607 OE2 GLU A 1128 −19.131 50.969 47.053 1.00 85.83 O ATOM 2608 N ALA A 1129 −22.587 53.372 42.561 1.00 67.55 N ATOM 2609 CA ALA A 1129 −23.000 54.375 41.574 1.00 71.98 C ATOM 2610 C ALA A 1129 −24.378 54.000 41.058 1.00 70.13 C ATOM 2611 O ALA A 1129 −25.263 54.827 41.057 1.00 76.48 O ATOM 2612 CB ALA A 1129 −21.989 54.462 40.410 1.00 65.05 C ATOM 2613 N ALA A 1130 −24.534 52.742 40.634 1.00 72.74 N ATOM 2614 CA ALA A 1130 −25.821 52.182 40.126 1.00 70.99 C ATOM 2615 C ALA A 1130 −26.956 52.249 41.142 1.00 70.01 C ATOM 2616 O ALA A 1130 −28.122 52.487 40.776 1.00 68.38 O ATOM 2617 CB ALA A 1130 −25.623 50.735 39.676 1.00 66.12 C ATOM 2618 N VAL A 1131 −26.632 52.039 42.416 1.00 68.58 N ATOM 2619 CA VAL A 1131 −27.663 52.124 43.473 1.00 67.85 C ATOM 2620 C VAL A 1131 −28.222 53.535 43.503 1.00 69.11 C ATOM 2621 O VAL A 1131 −29.445 53.719 43.551 1.00 67.62 O ATOM 2622 CB VAL A 1131 −27.149 51.685 44.851 1.00 69.41 C ATOM 2623 CG1 VAL A 1131 −28.070 52.236 46.024 1.00 60.58 C ATOM 2624 CG2 VAL A 1131 −27.016 50.147 44.886 1.00 64.52 C ATOM 2625 N ASN A 1132 −27.323 54.517 43.466 1.00 71.57 N ATOM 2626 CA ASN A 1132 −27.706 55.935 43.510 1.00 73.21 C ATOM 2627 C ASN A 1132 −28.408 56.412 42.232 1.00 70.74 C ATOM 2628 O ASN A 1132 −29.422 57.104 42.307 1.00 68.21 O ATOM 2629 CB ASN A 1132 −26.486 56.817 43.828 1.00 72.85 C ATOM 2630 CG ASN A 1132 −26.112 56.776 45.296 1.00 78.49 C ATOM 2631 OD1 ASN A 1132 −26.088 57.807 45.962 1.00 83.67 O ATOM 2632 ND2 ASN A 1132 −25.822 55.588 45.808 1.00 84.32 N ATOM 2633 N LEU A 1133 −27.869 56.040 41.076 1.00 69.80 N ATOM 2634 CA LEU A 1133 −28.465 56.421 39.798 1.00 72.25 C ATOM 2635 C LEU A 1133 −29.883 55.859 39.633 1.00 73.77 C ATOM 2636 O LEU A 1133 −30.748 56.528 39.065 1.00 78.51 O ATOM 2637 CB LEU A 1133 −27.569 55.971 38.637 1.00 74.37 C ATOM 2638 CG LEU A 1133 −26.186 56.632 38.565 1.00 74.55 C ATOM 2639 CD1 LEU A 1133 −25.327 56.045 37.436 1.00 81.95 C ATOM 2640 CD2 LEU A 1133 −26.336 58.139 38.408 1.00 79.93 C ATOM 2641 N ALA A 1134 −30.121 54.639 40.132 1.00 72.16 N ATOM 2642 CA ALA A 1134 −31.455 54.022 40.090 1.00 71.47 C ATOM 2643 C ALA A 1134 −32.505 54.776 40.950 1.00 72.41 C ATOM 2644 O ALA A 1134 −33.701 54.586 40.741 1.00 70.87 O ATOM 2645 CB ALA A 1134 −31.378 52.533 40.511 1.00 68.95 C ATOM 2646 N LYS A 1135 −32.052 55.620 41.901 1.00 68.74 N ATOM 2647 CA LYS A 1135 −32.946 56.436 42.773 1.00 72.99 C ATOM 2648 C LYS A 1135 −33.266 57.857 42.231 1.00 72.10 C ATOM 2649 O LYS A 1135 −33.585 58.762 43.000 1.00 74.92 O ATOM 2650 CB LYS A 1135 −32.343 56.536 44.199 1.00 69.32 C ATOM 2651 CG LYS A 1135 −32.402 55.253 45.012 1.00 76.21 C ATOM 2652 CD LYS A 1135 −31.674 55.410 46.367 1.00 77.13 C ATOM 2653 CE LYS A 1135 −31.950 54.245 47.310 1.00 77.91 C ATOM 2654 NZ LYS A 1135 −30.938 54.134 48.434 1.00 77.31 N ATOM 2655 N SER A 1136 −33.185 58.051 40.916 1.00 76.65 N ATOM 2656 CA SER A 1136 −33.404 59.364 40.298 1.00 74.00 C ATOM 2657 C SER A 1136 −34.753 59.516 39.594 1.00 76.99 C ATOM 2658 O SER A 1136 −35.464 58.536 39.320 1.00 79.35 O ATOM 2659 CB SER A 1136 −32.289 59.635 39.304 1.00 75.82 C ATOM 2660 OG SER A 1136 −32.139 58.573 38.368 1.00 74.98 O ATOM 2661 N ARG A 1137 −35.081 60.775 39.313 1.00 74.80 N ATOM 2662 CA ARG A 1137 −36.216 61.141 38.485 1.00 72.91 C ATOM 2663 C ARG A 1137 −35.965 60.639 37.043 1.00 71.62 C ATOM 2664 O ARG A 1137 −36.884 60.312 36.325 1.00 70.32 O ATOM 2665 CB ARG A 1137 −36.395 62.661 38.504 1.00 71.58 C ATOM 2666 CG ARG A 1137 −37.567 63.220 37.702 1.00 76.14 C ATOM 2667 CD ARG A 1137 −38.896 62.715 38.231 1.00 84.99 C ATOM 2668 NE ARG A 1137 −40.034 63.267 37.497 1.00 85.80 N ATOM 2669 CZ ARG A 1137 −41.305 62.894 37.664 1.00 83.56 C ATOM 2670 NH1 ARG A 1137 −41.640 61.951 38.547 1.00 74.66 N ATOM 2671 NH2 ARG A 1137 −42.263 63.469 36.933 1.00 88.55 N ATOM 2672 N TRP A 1138 −34.703 60.592 36.651 1.00 70.67 N ATOM 2673 CA TRP A 1138 −34.289 60.023 35.392 1.00 72.64 C ATOM 2674 C TRP A 1138 −34.759 58.569 35.248 1.00 74.44 C ATOM 2675 O TRP A 1138 −35.428 58.233 34.279 1.00 74.91 O ATOM 2676 CB TRP A 1138 −32.778 60.118 35.298 1.00 72.63 C ATOM 2677 CG TRP A 1138 −32.166 59.434 34.164 1.00 72.19 C ATOM 2678 CD1 TRP A 1138 −32.556 59.491 32.862 1.00 72.21 C ATOM 2679 CD2 TRP A 1138 −31.024 58.574 34.207 1.00 68.92 C ATOM 2680 NE1 TRP A 1138 −31.724 58.713 32.092 1.00 73.58 N ATOM 2681 CE2 TRP A 1138 −30.780 58.139 32.887 1.00 67.95 C ATOM 2682 CE3 TRP A 1138 −30.190 58.125 35.223 1.00 66.16 C ATOM 2683 CZ2 TRP A 1138 −29.749 57.293 32.570 1.00 69.41 C ATOM 2684 CZ3 TRP A 1138 −29.142 57.262 34.893 1.00 71.01 C ATOM 2685 CH2 TRP A 1138 −28.934 56.863 33.600 1.00 71.24 C ATOM 2686 N TYR A 1139 −34.407 57.727 36.212 1.00 73.44 N ATOM 2687 CA TYR A 1139 −34.828 56.318 36.196 1.00 70.88 C ATOM 2688 C TYR A 1139 −36.337 56.170 36.397 1.00 71.38 C ATOM 2689 O TYR A 1139 −36.940 55.218 35.922 1.00 72.03 O ATOM 2690 CB TYR A 1139 −34.064 55.516 37.258 1.00 74.06 C ATOM 2691 CG TYR A 1139 −34.530 54.095 37.404 1.00 70.82 C ATOM 2692 CD1 TYR A 1139 −34.012 53.101 36.601 1.00 76.39 C ATOM 2693 CD2 TYR A 1139 −35.496 53.746 38.351 1.00 76.13 C ATOM 2694 CE1 TYR A 1139 −34.429 51.793 36.720 1.00 73.02 C ATOM 2695 CE2 TYR A 1139 −35.923 52.436 38.481 1.00 79.81 C ATOM 2696 CZ TYR A 1139 −35.376 51.461 37.651 1.00 77.76 C ATOM 2697 OH TYR A 1139 −35.772 50.154 37.747 1.00 78.31 O ATOM 2698 N ASN A 1140 −36.943 57.111 37.106 1.00 72.72 N ATOM 2699 CA ASN A 1140 −38.381 57.111 37.312 1.00 71.50 C ATOM 2700 C ASN A 1140 −39.175 57.391 36.028 1.00 70.46 C ATOM 2701 O ASN A 1140 −40.307 56.929 35.882 1.00 72.15 O ATOM 2702 CB ASN A 1140 −38.755 58.172 38.338 1.00 68.60 C ATOM 2703 CG ASN A 1140 −40.230 58.240 38.572 1.00 72.49 C ATOM 2704 OD1 ASN A 1140 −40.864 59.264 38.335 1.00 72.97 O ATOM 2705 ND2 ASN A 1140 −40.801 57.138 39.040 1.00 83.45 N ATOM 2706 N GLN A 1141 −38.573 58.145 35.111 1.00 71.15 N ATOM 2707 CA GLN A 1141 −39.248 58.616 33.896 1.00 71.46 C ATOM 2708 C GLN A 1141 −38.919 57.755 32.677 1.00 72.46 C ATOM 2709 O GLN A 1141 −39.828 57.378 31.930 1.00 69.45 O ATOM 2710 CB GLN A 1141 −38.887 60.091 33.663 1.00 71.54 C ATOM 2711 CG GLN A 1141 −39.493 61.040 34.694 1.00 74.06 C ATOM 2712 CD GLN A 1141 −40.987 61.142 34.576 1.00 84.66 C ATOM 2713 OE1 GLN A 1141 −41.726 60.867 35.539 1.00 72.30 O ATOM 2714 NE2 GLN A 1141 −41.461 61.535 33.387 1.00 78.96 N ATOM 2715 N THR A 1142 −37.633 57.454 32.490 1.00 69.68 N ATOM 2716 CA THR A 1142 −37.167 56.582 31.409 1.00 71.24 C ATOM 2717 C THR A 1142 −36.399 55.382 31.997 1.00 71.57 C ATOM 2718 O THR A 1142 −35.177 55.274 31.783 1.00 73.67 O ATOM 2719 CB THR A 1142 −36.318 57.373 30.396 1.00 73.10 C ATOM 2720 OG1 THR A 1142 −35.335 58.134 31.100 1.00 65.73 O ATOM 2721 CG2 THR A 1142 −37.223 58.326 29.569 1.00 63.92 C ATOM 2722 N PRO A 1143 −37.120 54.475 32.744 1.00 68.91 N ATOM 2723 CA PRO A 1143 −36.537 53.299 33.404 1.00 67.95 C ATOM 2724 C PRO A 1143 −35.762 52.372 32.497 1.00 71.67 C ATOM 2725 O PRO A 1143 −34.638 52.018 32.834 1.00 70.47 O ATOM 2726 CB PRO A 1143 −37.760 52.553 33.937 1.00 70.48 C ATOM 2727 CG PRO A 1143 −38.920 53.137 33.252 1.00 70.91 C ATOM 2728 CD PRO A 1143 −38.570 54.528 33.001 1.00 68.98 C ATOM 2729 N ASN A 1144 −36.357 51.993 31.361 1.00 70.87 N ATOM 2730 CA ASN A 1144 −35.706 51.083 30.410 1.00 71.14 C ATOM 2731 C ASN A 1144 −34.382 51.613 29.946 1.00 72.08 C ATOM 2732 O ASN A 1144 −33.369 50.921 30.071 1.00 71.77 O ATOM 2733 CB ASN A 1144 −36.582 50.818 29.180 1.00 71.11 C ATOM 2734 CG ASN A 1144 −37.859 50.077 29.515 1.00 74.27 C ATOM 2735 OD1 ASN A 1144 −37.919 49.323 30.480 1.00 76.06 O ATOM 2736 ND2 ASN A 1144 −38.895 50.288 28.709 1.00 86.76 N ATOM 2737 N ARG A 1145 −34.388 52.837 29.409 1.00 70.29 N ATOM 2738 CA ARG A 1145 −33.147 53.500 28.979 1.00 69.78 C ATOM 2739 C ARG A 1145 −32.142 53.627 30.132 1.00 70.79 C ATOM 2740 O ARG A 1145 −30.950 53.299 29.984 1.00 71.02 O ATOM 2741 CB ARG A 1145 −33.450 54.880 28.462 1.00 69.40 C ATOM 2742 CG ARG A 1145 −32.215 55.697 28.103 1.00 72.46 C ATOM 2743 CD ARG A 1145 −32.619 56.971 27.368 1.00 71.18 C ATOM 2744 NE ARG A 1145 −31.446 57.732 26.931 1.00 74.68 N ATOM 2745 CZ ARG A 1145 −31.488 58.840 26.186 1.00 81.63 C ATOM 2746 NH1 ARG A 1145 −32.661 59.353 25.769 1.00 76.56 N ATOM 2747 NH2 ARG A 1145 −30.354 59.453 25.844 1.00 80.74 N ATOM 2748 N ALA A 1146 −32.632 54.105 31.272 1.00 68.56 N ATOM 2749 CA ALA A 1146 −31.782 54.265 32.437 1.00 70.12 C ATOM 2750 C ALA A 1146 −31.211 52.906 32.844 1.00 68.68 C ATOM 2751 O ALA A 1146 −30.039 52.815 33.194 1.00 70.80 O ATOM 2752 CB ALA A 1146 −32.534 54.894 33.547 1.00 66.71 C ATOM 2753 N LYS A 1147 −32.029 51.862 32.786 1.00 69.92 N ATOM 2754 CA LYS A 1147 −31.583 50.524 33.161 1.00 72.10 C ATOM 2755 C LYS A 1147 −30.365 50.177 32.322 1.00 72.04 C ATOM 2756 O LYS A 1147 −29.317 49.794 32.881 1.00 66.93 O ATOM 2757 CB LYS A 1147 −32.712 49.514 32.992 1.00 73.45 C ATOM 2758 CG LYS A 1147 −32.536 48.177 33.665 1.00 78.51 C ATOM 2759 CD LYS A 1147 −33.835 47.348 33.477 1.00 82.18 C ATOM 2760 CE LYS A 1147 −33.762 45.956 34.103 1.00 93.88 C ATOM 2761 NZ LYS A 1147 −33.816 45.967 35.617 1.00 96.78 N ATOM 2762 N ARG A 1148 −30.494 50.323 30.986 1.00 73.00 N ATOM 2763 CA ARG A 1148 −29.393 50.016 30.051 1.00 70.89 C ATOM 2764 C ARG A 1148 −28.135 50.860 30.239 1.00 74.78 C ATOM 2765 O ARG A 1148 −27.034 50.334 30.098 1.00 80.02 O ATOM 2766 CB ARG A 1148 −29.832 50.150 28.590 1.00 72.03 C ATOM 2767 CG ARG A 1148 −30.780 49.068 28.099 1.00 70.33 C ATOM 2768 CD ARG A 1148 −30.936 49.102 26.596 1.00 72.06 C ATOM 2769 NE ARG A 1148 −31.674 50.290 26.158 1.00 71.65 N ATOM 2770 CZ ARG A 1148 −33.006 50.391 26.084 1.00 72.14 C ATOM 2771 NH1 ARG A 1148 −33.801 49.382 26.416 1.00 78.11 N ATOM 2772 NH2 ARG A 1148 −33.568 51.523 25.671 1.00 71.64 N ATOM 2773 N VAL A 1149 −28.270 52.150 30.547 1.00 74.24 N ATOM 2774 CA VAL A 1149 −27.072 52.990 30.752 1.00 71.90 C ATOM 2775 C VAL A 1149 −26.399 52.632 32.098 1.00 74.21 C ATOM 2776 O VAL A 1149 −25.188 52.478 32.164 1.00 77.27 O ATOM 2777 CB VAL A 1149 −27.374 54.510 30.676 1.00 78.88 C ATOM 2778 CG1 VAL A 1149 −26.122 55.316 31.006 1.00 78.08 C ATOM 2779 CG2 VAL A 1149 −27.939 54.922 29.282 1.00 63.48 C ATOM 2780 N ILE A 1150 −27.182 52.498 33.158 1.00 70.73 N ATOM 2781 CA ILE A 1150 −26.623 52.164 34.467 1.00 70.91 C ATOM 2782 C ILE A 1150 −25.901 50.823 34.437 1.00 69.25 C ATOM 2783 O ILE A 1150 −24.744 50.727 34.864 1.00 74.32 O ATOM 2784 CB ILE A 1150 −27.689 52.145 35.570 1.00 71.99 C ATOM 2785 CG1 ILE A 1150 −28.264 53.541 35.793 1.00 69.99 C ATOM 2786 CG2 ILE A 1150 −27.077 51.645 36.848 1.00 70.14 C ATOM 2787 CD1 ILE A 1150 −29.550 53.539 36.637 1.00 76.32 C ATOM 2788 N THR A 1151 −26.578 49.793 33.938 1.00 67.61 N ATOM 2789 CA THR A 1151 −25.954 48.476 33.707 1.00 67.20 C ATOM 2790 C THR A 1151 −24.648 48.608 32.920 1.00 70.88 C ATOM 2791 O THR A 1151 −23.683 47.887 33.190 1.00 74.26 O ATOM 2792 CB THR A 1151 −26.876 47.558 32.905 1.00 68.20 C ATOM 2793 OG1 THR A 1151 −28.158 47.491 33.541 1.00 72.09 O ATOM 2794 CG2 THR A 1151 −26.270 46.158 32.776 1.00 64.27 C ATOM 2795 N THR A 1152 −24.619 49.527 31.941 1.00 67.75 N ATOM 2796 CA THR A 1152 −23.399 49.788 31.157 1.00 70.23 C ATOM 2797 C THR A 1152 −22.322 50.417 32.042 1.00 69.27 C ATOM 2798 O THR A 1152 −21.145 50.068 31.924 1.00 71.67 O ATOM 2799 CB THR A 1152 −23.676 50.696 29.941 1.00 66.28 C ATOM 2800 OG1 THR A 1152 −24.680 50.076 29.121 1.00 70.58 O ATOM 2801 CG2 THR A 1152 −22.396 50.939 29.157 1.00 69.86 C ATOM 2802 N PHE A 1153 −22.719 51.337 32.913 1.00 68.71 N ATOM 2803 CA PHE A 1153 −21.791 51.796 33.990 1.00 74.06 C ATOM 2804 C PHE A 1153 −21.345 50.649 34.890 1.00 70.59 C ATOM 2805 O PHE A 1153 −20.232 50.662 35.389 1.00 76.73 O ATOM 2806 CB PHE A 1153 −22.391 52.892 34.872 1.00 71.58 C ATOM 2807 CG PHE A 1153 −22.359 54.274 34.258 1.00 75.33 C ATOM 2808 CD1 PHE A 1153 −21.180 54.807 33.778 1.00 76.55 C ATOM 2809 CD2 PHE A 1153 −23.508 55.034 34.170 1.00 79.26 C ATOM 2810 CE1 PHE A 1153 −21.147 56.059 33.224 1.00 73.15 C ATOM 2811 CE2 PHE A 1153 −23.479 56.282 33.622 1.00 72.03 C ATOM 2812 CZ PHE A 1153 −22.296 56.799 33.147 1.00 71.84 C ATOM 2813 N ARG A 1154 −22.198 49.655 35.108 1.00 70.31 N ATOM 2814 CA ARG A 1154 −21.801 48.490 35.940 1.00 72.56 C ATOM 2815 C ARG A 1154 −20.774 47.559 35.312 1.00 74.07 C ATOM 2816 O ARG A 1154 −19.826 47.158 35.994 1.00 75.46 O ATOM 2817 CB ARG A 1154 −23.013 47.661 36.354 1.00 71.60 C ATOM 2818 CG ARG A 1154 −23.926 48.392 37.315 1.00 78.93 C ATOM 2819 CD ARG A 1154 −24.313 47.484 38.485 1.00 80.49 C ATOM 2820 NE ARG A 1154 −25.312 46.492 38.126 1.00 81.98 N ATOM 2821 CZ ARG A 1154 −25.657 45.436 38.870 1.00 84.48 C ATOM 2822 NH1 ARG A 1154 −25.082 45.186 40.063 1.00 90.99 N ATOM 2823 NH2 ARG A 1154 −26.595 44.601 38.421 1.00 88.13 N ATOM 2824 N THR A 1155 −20.956 47.219 34.029 1.00 73.12 N ATOM 2825 CA THR A 1155 −20.095 46.232 33.346 1.00 74.07 C ATOM 2826 C THR A 1155 −18.969 46.828 32.501 1.00 76.10 C ATOM 2827 O THR A 1155 −17.912 46.218 32.361 1.00 76.98 O ATOM 2828 CB THR A 1155 −20.940 45.324 32.422 1.00 75.54 C ATOM 2829 OG1 THR A 1155 −21.368 46.063 31.251 1.00 76.27 O ATOM 2830 CG2 THR A 1155 −22.152 44.777 33.176 1.00 73.31 C ATOM 2831 N GLY A 1156 −19.185 48.008 31.936 1.00 75.88 N ATOM 2832 CA GLY A 1156 −18.198 48.591 31.038 1.00 76.99 C ATOM 2833 C GLY A 1156 −18.113 47.829 29.703 1.00 78.89 C ATOM 2834 O GLY A 1156 −17.054 47.785 29.073 1.00 77.75 O ATOM 2835 N THR A 1157 −19.240 47.235 29.297 1.00 79.45 N ATOM 2836 CA THR A 1157 −19.388 46.573 28.025 1.00 75.30 C ATOM 2837 C THR A 1157 −20.713 47.007 27.435 1.00 76.85 C ATOM 2838 O THR A 1157 −21.568 47.591 28.121 1.00 72.71 O ATOM 2839 CB THR A 1157 −19.491 45.069 28.150 1.00 77.35 C ATOM 2840 OG1 THR A 1157 −20.771 44.747 28.718 1.00 78.06 O ATOM 2841 CG2 THR A 1157 −18.332 44.460 29.016 1.00 73.49 C ATOM 2842 N TRP A 1158 −20.880 46.706 26.149 1.00 75.57 N ATOM 2843 CA TRP A 1158 −22.102 47.000 25.412 1.00 72.41 C ATOM 2844 C TRP A 1158 −23.213 45.956 25.579 1.00 72.74 C ATOM 2845 O TRP A 1158 −24.243 46.070 24.904 1.00 74.46 O ATOM 2846 CB TRP A 1158 −21.769 47.101 23.927 1.00 76.07 C ATOM 2847 CG TRP A 1158 −20.844 48.147 23.646 1.00 74.03 C ATOM 2848 CD1 TRP A 1158 −19.563 48.007 23.274 1.00 71.12 C ATOM 2849 CD2 TRP A 1158 −21.107 49.539 23.717 1.00 72.83 C ATOM 2850 NE1 TRP A 1158 −18.986 49.238 23.096 1.00 76.80 N ATOM 2851 CE2 TRP A 1158 −19.916 50.200 23.362 1.00 66.00 C ATOM 2852 CE3 TRP A 1158 −22.238 50.297 24.049 1.00 74.60 C ATOM 2853 CZ2 TRP A 1158 −19.811 51.582 23.322 1.00 71.92 C ATOM 2854 CZ3 TRP A 1158 −22.138 51.673 24.012 1.00 76.31 C ATOM 2855 CH2 TRP A 1158 −20.927 52.307 23.649 1.00 74.45 C ATOM 2856 N ASP A 1159 −23.019 44.955 26.460 1.00 72.02 N ATOM 2857 CA ASP A 1159 −23.967 43.835 26.664 1.00 71.28 C ATOM 2858 C ASP A 1159 −25.450 44.207 26.837 1.00 72.00 C ATOM 2859 O ASP A 1159 −26.316 43.422 26.450 1.00 73.98 O ATOM 2860 CB ASP A 1159 −23.527 42.966 27.852 1.00 73.56 C ATOM 2861 CG ASP A 1159 −22.254 42.143 27.570 1.00 78.31 C ATOM 2862 OD1 ASP A 1159 −21.715 42.178 26.450 1.00 78.77 O ATOM 2863 OD2 ASP A 1159 −21.791 41.449 28.499 1.00 92.78 O ATOM 2864 N ALA A 1160 −25.746 45.381 27.405 1.00 72.33 N ATOM 2865 CA ALA A 1160 −27.154 45.828 27.567 1.00 72.44 C ATOM 2866 C ALA A 1160 −27.815 46.215 26.238 1.00 72.65 C ATOM 2867 O ALA A 1160 −29.037 46.285 26.169 1.00 75.90 O ATOM 2868 CB ALA A 1160 −27.254 46.998 28.560 1.00 69.43 C ATOM 2869 N TYR A 1161 −27.010 46.465 25.200 1.00 73.56 N ATOM 2870 CA TYR A 1161 −27.500 46.850 23.870 1.00 70.85 C ATOM 2871 C TYR A 1161 −27.500 45.693 22.845 1.00 71.12 C ATOM 2872 O TYR A 1161 −27.779 45.928 21.684 1.00 74.87 O ATOM 2873 CB TYR A 1161 −26.712 48.067 23.381 1.00 70.71 C ATOM 2874 CG TYR A 1161 −26.977 49.224 24.282 1.00 69.57 C ATOM 2875 CD1 TYR A 1161 −28.020 50.090 24.037 1.00 67.53 C ATOM 2876 CD2 TYR A 1161 −26.187 49.451 25.400 1.00 71.83 C ATOM 2877 CE1 TYR A 1161 −28.269 51.157 24.878 1.00 69.80 C ATOM 2878 CE2 TYR A 1161 −26.432 50.509 26.236 1.00 64.36 C ATOM 2879 CZ TYR A 1161 −27.470 51.366 25.980 1.00 73.12 C ATOM 2880 OH TYR A 1161 −27.695 52.436 26.850 1.00 77.23 O ATOM 2881 N LYS A 263 −27.193 44.468 23.289 1.00 68.62 N ATOM 2882 CA LYS A 263 −27.211 43.302 22.437 1.00 68.39 C ATOM 2883 C LYS A 263 −28.617 42.992 22.039 1.00 68.68 C ATOM 2884 O LYS A 263 −29.497 43.032 22.873 1.00 71.28 O ATOM 2885 CB LYS A 263 −26.716 42.052 23.152 1.00 70.24 C ATOM 2886 CG LYS A 263 −25.247 41.957 23.391 1.00 76.63 C ATOM 2887 CD LYS A 263 −24.916 40.567 23.966 1.00 77.14 C ATOM 2888 CE LYS A 263 −23.433 40.257 23.926 1.00 86.04 C ATOM 2889 NZ LYS A 263 −23.139 38.781 24.083 1.00 88.05 N ATOM 2890 N PHE A 264 −28.823 42.683 20.766 1.00 66.31 N ATOM 2891 CA PHE A 264 −30.129 42.267 20.240 1.00 67.14 C ATOM 2892 C PHE A 264 −31.247 43.272 20.499 1.00 66.08 C ATOM 2893 O PHE A 264 −32.385 42.892 20.754 1.00 66.16 O ATOM 2894 CB PHE A 264 −30.490 40.877 20.774 1.00 65.00 C ATOM 2895 CG PHE A 264 −29.455 39.843 20.461 1.00 68.77 C ATOM 2896 CD1 PHE A 264 −28.761 39.182 21.465 1.00 69.39 C ATOM 2897 CD2 PHE A 264 −29.166 39.530 19.135 1.00 69.77 C ATOM 2898 CE1 PHE A 264 −27.800 38.223 21.148 1.00 67.15 C ATOM 2899 CE2 PHE A 264 −28.210 38.578 18.825 1.00 68.88 C ATOM 2900 CZ PHE A 264 −27.531 37.925 19.832 1.00 67.43 C ATOM 2901 N CYS A 265 −30.891 44.551 20.430 1.00 66.76 N ATOM 2902 CA CYS A 265 −31.837 45.636 20.494 1.00 66.33 C ATOM 2903 C CYS A 265 −32.192 46.034 19.051 1.00 66.21 C ATOM 2904 O CYS A 265 −33.355 46.217 18.742 1.00 67.25 O ATOM 2905 CB CYS A 265 −31.269 46.818 21.274 1.00 65.09 C ATOM 2906 SG CYS A 265 −31.066 46.521 23.110 1.00 69.25 S ATOM 2907 N LEU A 266 −31.183 46.163 18.186 1.00 63.62 N ATOM 2908 CA LEU A 266 −31.385 46.560 16.806 1.00 65.11 C ATOM 2909 C LEU A 266 −32.130 45.487 16.037 1.00 65.91 C ATOM 2910 O LEU A 266 −31.815 44.312 16.172 1.00 65.41 O ATOM 2911 CB LEU A 266 −30.044 46.787 16.116 1.00 63.47 C ATOM 2912 CG LEU A 266 −29.137 47.928 16.567 1.00 66.69 C ATOM 2913 CD1 LEU A 266 −27.791 47.827 15.848 1.00 64.50 C ATOM 2914 CD2 LEU A 266 −29.796 49.271 16.325 1.00 66.85 C ATOM 2915 N LYS A 267 −33.112 45.898 15.238 1.00 68.75 N ATOM 2916 CA LYS A 267 −33.870 44.973 14.350 1.00 71.90 C ATOM 2917 C LYS A 267 −32.973 44.007 13.566 1.00 69.09 C ATOM 2918 O LYS A 267 −33.274 42.829 13.463 1.00 72.39 O ATOM 2919 CB LYS A 267 −34.768 45.762 13.373 1.00 74.97 C ATOM 2920 CG LYS A 267 −34.008 46.721 12.419 1.00 82.81 C ATOM 2921 CD LYS A 267 −34.913 47.656 11.634 1.00 84.17 C ATOM 2922 CE LYS A 267 −34.071 48.658 10.816 1.00 88.72 C ATOM 2923 NZ LYS A 267 −34.904 49.777 10.290 1.00 88.47 N ATOM 2924 N GLU A 268 −31.877 44.519 13.024 1.00 68.14 N ATOM 2925 CA GLU A 268 −30.929 43.706 12.257 1.00 68.69 C ATOM 2926 C GLU A 268 −30.232 42.665 13.125 1.00 69.97 C ATOM 2927 O GLU A 268 −30.010 41.554 12.679 1.00 71.10 O ATOM 2928 CB GLU A 268 −29.897 44.568 11.492 1.00 71.25 C ATOM 2929 CG GLU A 268 −29.063 45.591 12.318 1.00 82.60 C ATOM 2930 CD GLU A 268 −29.720 46.975 12.491 1.00 84.41 C ATOM 2931 OE1 GLU A 268 −30.934 47.164 12.219 1.00 81.45 O ATOM 2932 OE2 GLU A 268 −28.992 47.877 12.911 1.00 83.78 O ATOM 2933 N HIS A 269 −29.891 43.023 14.366 1.00 70.32 N ATOM 2934 CA HIS A 269 −29.274 42.065 15.287 1.00 68.22 C ATOM 2935 C HIS A 269 −30.290 41.039 15.757 1.00 66.82 C ATOM 2936 O HIS A 269 −29.946 39.856 15.882 1.00 65.02 O ATOM 2937 CB HIS A 269 −28.612 42.794 16.451 1.00 71.92 C ATOM 2938 CG HIS A 269 −27.390 43.554 16.047 1.00 71.60 C ATOM 2939 ND1 HIS A 269 −26.547 44.134 16.958 1.00 68.24 N ATOM 2940 CD2 HIS A 269 −26.867 43.824 14.824 1.00 75.13 C ATOM 2941 CE1 HIS A 269 −25.557 44.734 16.325 1.00 73.61 C ATOM 2942 NE2 HIS A 269 −25.725 44.562 15.027 1.00 78.78 N ATOM 2943 N LYS A 270 −31.526 41.477 16.014 1.00 64.48 N ATOM 2944 CA LYS A 270 −32.619 40.536 16.296 1.00 67.98 C ATOM 2945 C LYS A 270 −32.761 39.521 15.146 1.00 68.82 C ATOM 2946 O LYS A 270 −32.861 38.328 15.394 1.00 66.36 O ATOM 2947 CB LYS A 270 −33.948 41.247 16.519 1.00 66.79 C ATOM 2948 CG LYS A 270 −33.987 42.127 17.773 1.00 73.23 C ATOM 2949 CD LYS A 270 −35.412 42.556 18.088 1.00 71.51 C ATOM 2950 CE LYS A 270 −35.487 43.405 19.345 1.00 75.87 C ATOM 2951 NZ LYS A 270 −35.017 42.669 20.545 1.00 77.55 N ATOM 2952 N ALA A 271 −32.764 40.009 13.897 1.00 69.99 N ATOM 2953 CA ALA A 271 −32.855 39.121 12.713 1.00 71.38 C ATOM 2954 C ALA A 271 −31.731 38.111 12.727 1.00 71.12 C ATOM 2955 O ALA A 271 −31.975 36.946 12.466 1.00 73.66 O ATOM 2956 CB ALA A 271 −32.855 39.918 11.399 1.00 64.88 C ATOM 2957 N LEU A 272 −30.502 38.548 13.034 1.00 72.07 N ATOM 2958 CA LEU A 272 −29.359 37.599 13.151 1.00 74.58 C ATOM 2959 C LEU A 272 −29.561 36.513 14.240 1.00 75.85 C ATOM 2960 O LEU A 272 −29.137 35.367 14.051 1.00 81.93 O ATOM 2961 CB LEU A 272 −28.025 38.324 13.408 1.00 73.91 C ATOM 2962 CG LEU A 272 −27.406 39.228 12.329 1.00 78.65 C ATOM 2963 CD1 LEU A 272 −26.129 39.864 12.873 1.00 79.09 C ATOM 2964 CD2 LEU A 272 −27.118 38.465 11.080 1.00 65.55 C ATOM 2965 N LYS A 273 −30.195 36.864 15.358 1.00 76.42 N ATOM 2966 CA LYS A 273 −30.509 35.866 16.403 1.00 77.14 C ATOM 2967 C LYS A 273 −31.512 34.845 15.857 1.00 77.91 C ATOM 2968 O LYS A 273 −31.346 33.663 16.083 1.00 76.67 O ATOM 2969 CB LYS A 273 −31.049 36.529 17.656 1.00 78.11 C ATOM 2970 CG LYS A 273 −31.226 35.608 18.867 1.00 80.19 C ATOM 2971 CD LYS A 273 −31.473 36.443 20.100 1.00 79.05 C ATOM 2972 CE LYS A 273 −31.752 35.624 21.329 1.00 82.30 C ATOM 2973 NZ LYS A 273 −31.953 36.544 22.492 1.00 84.12 N ATOM 2974 N THR A 274 −32.541 35.313 15.139 1.00 80.30 N ATOM 2975 CA THR A 274 −33.525 34.414 14.487 1.00 78.23 C ATOM 2976 C THR A 274 −32.827 33.369 13.651 1.00 76.51 C ATOM 2977 O THR A 274 −33.082 32.191 13.826 1.00 75.03 O ATOM 2978 CB THR A 274 −34.520 35.166 13.564 1.00 81.00 C ATOM 2979 OG1 THR A 274 −35.198 36.197 14.293 1.00 83.35 O ATOM 2980 CG2 THR A 274 −35.553 34.206 12.963 1.00 79.62 C ATOM 2981 N LEU A 275 −31.950 33.800 12.754 1.00 73.92 N ATOM 2982 CA LEU A 275 −31.183 32.862 11.925 1.00 74.39 C ATOM 2983 C LEU A 275 −30.330 31.927 12.779 1.00 75.45 C ATOM 2984 O LEU A 275 −30.279 30.721 12.524 1.00 76.13 O ATOM 2985 CB LEU A 275 −30.301 33.605 10.922 1.00 75.70 C ATOM 2986 CG LEU A 275 −30.975 34.417 9.808 1.00 75.16 C ATOM 2987 CD1 LEU A 275 −29.909 35.169 8.981 1.00 67.26 C ATOM 2988 CD2 LEU A 275 −31.846 33.533 8.910 1.00 70.95 C ATOM 2989 N GLY A 276 −29.663 32.476 13.797 1.00 78.78 N ATOM 2990 CA GLY A 276 −28.890 31.661 14.740 1.00 73.44 C ATOM 2991 C GLY A 276 −29.740 30.605 15.422 1.00 73.61 C ATOM 2992 O GLY A 276 −29.247 29.515 15.743 1.00 74.08 O ATOM 2993 N ILE A 277 −31.017 30.921 15.645 1.00 73.26 N ATOM 2994 CA ILE A 277 −31.955 29.987 16.286 1.00 76.95 C ATOM 2995 C ILE A 277 −32.354 28.862 15.334 1.00 75.85 C ATOM 2996 O ILE A 277 −32.540 27.720 15.768 1.00 77.29 O ATOM 2997 CB ILE A 277 −33.202 30.721 16.866 1.00 74.67 C ATOM 2998 CG1 ILE A 277 −32.797 31.558 18.081 1.00 76.85 C ATOM 2999 CG2 ILE A 277 −34.275 29.747 17.287 1.00 72.00 C ATOM 3000 CD1 ILE A 277 −33.970 32.239 18.788 1.00 76.44 C ATOM 3001 N ILE A 278 −32.481 29.188 14.048 1.00 79.83 N ATOM 3002 CA ILE A 278 −32.787 28.208 13.010 1.00 77.27 C ATOM 3003 C ILE A 278 −31.630 27.225 12.931 1.00 79.77 C ATOM 3004 O ILE A 278 −31.833 26.004 12.877 1.00 80.80 O ATOM 3005 CB ILE A 278 −33.004 28.892 11.637 1.00 77.96 C ATOM 3006 CG1 ILE A 278 −34.244 29.798 11.651 1.00 79.43 C ATOM 3007 CG2 ILE A 278 −33.174 27.888 10.557 1.00 78.57 C ATOM 3008 CD1 ILE A 278 −35.544 29.087 11.775 1.00 83.47 C ATOM 3009 N MET A 279 −30.412 27.761 12.933 1.00 79.21 N ATOM 3010 CA MET A 279 −29.206 26.942 12.826 1.00 77.35 C ATOM 3011 C MET A 279 −29.019 26.044 14.018 1.00 75.85 C ATOM 3012 O MET A 279 −28.798 24.844 13.852 1.00 73.53 O ATOM 3013 CB MET A 279 −27.982 27.830 12.632 1.00 76.42 C ATOM 3014 CG MET A 279 −27.992 28.535 11.284 1.00 79.03 C ATOM 3015 SD MET A 279 −26.852 29.897 11.156 1.00 80.66 S ATOM 3016 CE MET A 279 −27.061 30.304 9.407 1.00 79.86 C ATOM 3017 N GLY A 280 −29.112 26.625 15.214 1.00 78.82 N ATOM 3018 CA GLY A 280 −28.937 25.880 16.463 1.00 76.24 C ATOM 3019 C GLY A 280 −29.970 24.785 16.660 1.00 79.31 C ATOM 3020 O GLY A 280 −29.640 23.694 17.120 1.00 80.01 O ATOM 3021 N THR A 281 −31.219 25.084 16.309 1.00 80.59 N ATOM 3022 CA THR A 281 −32.309 24.123 16.408 1.00 78.83 C ATOM 3023 C THR A 281 −32.057 22.935 15.492 1.00 77.28 C ATOM 3024 O THR A 281 −32.190 21.801 15.923 1.00 76.07 O ATOM 3025 CB THR A 281 −33.657 24.780 16.070 1.00 81.71 C ATOM 3026 OG1 THR A 281 −33.964 25.771 17.065 1.00 79.40 O ATOM 3027 CG2 THR A 281 −34.769 23.760 16.038 1.00 82.36 C ATOM 3028 N PHE A 282 −31.694 23.205 14.237 1.00 79.67 N ATOM 3029 CA PHE A 282 −31.347 22.147 13.279 1.00 77.00 C ATOM 3030 C PHE A 282 −30.221 21.282 13.817 1.00 76.25 C ATOM 3031 O PHE A 282 −30.266 20.069 13.697 1.00 73.80 O ATOM 3032 CB PHE A 282 −30.917 22.731 11.930 1.00 77.95 C ATOM 3033 CG PHE A 282 −30.691 21.679 10.853 1.00 76.73 C ATOM 3034 CD1 PHE A 282 −31.723 21.314 9.995 1.00 75.49 C ATOM 3035 CD2 PHE A 282 −29.460 21.064 10.706 1.00 76.37 C ATOM 3036 CE1 PHE A 282 −31.526 20.343 8.997 1.00 76.36 C ATOM 3037 CE2 PHE A 282 −29.254 20.097 9.720 1.00 84.43 C ATOM 3038 CZ PHE A 282 −30.297 19.737 8.857 1.00 76.69 C ATOM 3039 N THR A 283 −29.214 21.923 14.406 1.00 77.11 N ATOM 3040 CA THR A 283 −28.058 21.218 14.945 1.00 76.40 C ATOM 3041 C THR A 283 −28.460 20.311 16.078 1.00 76.62 C ATOM 3042 O THR A 283 −28.105 19.152 16.073 1.00 76.69 O ATOM 3043 CB THR A 283 −26.981 22.183 15.448 1.00 76.06 C ATOM 3044 OG1 THR A 283 −26.544 23.023 14.367 1.00 80.64 O ATOM 3045 CG2 THR A 283 −25.784 21.412 16.018 1.00 75.59 C ATOM 3046 N LEU A 284 −29.203 20.844 17.041 1.00 78.59 N ATOM 3047 CA LEU A 284 −29.636 20.065 18.204 1.00 77.27 C ATOM 3048 C LEU A 284 −30.578 18.920 17.845 1.00 77.57 C ATOM 3049 O LEU A 284 −30.560 17.886 18.500 1.00 76.25 O ATOM 3050 CB LEU A 284 −30.300 20.986 19.236 1.00 80.83 C ATOM 3051 CG LEU A 284 −29.359 22.013 19.916 1.00 89.13 C ATOM 3052 CD1 LEU A 284 −30.162 23.029 20.724 1.00 93.20 C ATOM 3053 CD2 LEU A 284 −28.290 21.337 20.801 1.00 91.45 C ATOM 3054 N CYS A 285 −31.395 19.108 16.806 1.00 77.86 N ATOM 3055 CA CYS A 285 −32.341 18.073 16.367 1.00 76.99 C ATOM 3056 C CYS A 285 −31.677 16.920 15.620 1.00 75.92 C ATOM 3057 O CYS A 285 −32.095 15.782 15.783 1.00 75.32 O ATOM 3058 CB CYS A 285 −33.462 18.678 15.513 1.00 76.74 C ATOM 3059 SG CYS A 285 −34.727 19.541 16.511 1.00 86.35 S ATOM 3060 N TRP A 286 −30.657 17.219 14.810 1.00 76.29 N ATOM 3061 CA TRP A 286 −29.938 16.192 14.020 1.00 75.01 C ATOM 3062 C TRP A 286 −28.635 15.671 14.635 1.00 75.15 C ATOM 3063 O TRP A 286 −28.250 14.544 14.330 1.00 75.31 O ATOM 3064 CB TRP A 286 −29.641 16.703 12.599 1.00 72.75 C ATOM 3065 CG TRP A 286 −30.853 16.738 11.766 1.00 70.37 C ATOM 3066 CD1 TRP A 286 −31.543 17.832 11.381 1.00 69.67 C ATOM 3067 CD2 TRP A 286 −31.533 15.613 11.212 1.00 67.81 C ATOM 3068 NE1 TRP A 286 −32.621 17.469 10.614 1.00 76.08 N ATOM 3069 CE2 TRP A 286 −32.636 16.107 10.496 1.00 67.36 C ATOM 3070 CE3 TRP A 286 −31.314 14.228 11.254 1.00 69.38 C ATOM 3071 CZ2 TRP A 286 −33.523 15.274 9.822 1.00 73.52 C ATOM 3072 CZ3 TRP A 286 −32.202 13.391 10.581 1.00 71.27 C ATOM 3073 CH2 TRP A 286 −33.291 13.920 9.875 1.00 72.45 C ATOM 3074 N LEU A 287 −27.958 16.454 15.474 1.00 71.19 N ATOM 3075 CA LEU A 287 −26.645 16.020 16.001 1.00 72.17 C ATOM 3076 C LEU A 287 −26.669 14.651 16.667 1.00 70.61 C ATOM 3077 O LEU A 287 −25.822 13.834 16.347 1.00 73.46 O ATOM 3078 CB LEU A 287 −26.019 17.049 16.953 1.00 74.35 C ATOM 3079 CG LEU A 287 −24.542 16.884 17.277 1.00 76.88 C ATOM 3080 CD1 LEU A 287 −23.682 16.964 16.013 1.00 78.66 C ATOM 3081 CD2 LEU A 287 −24.130 17.956 18.279 1.00 79.06 C ATOM 3082 N PRO A 288 −27.627 14.395 17.583 1.00 69.58 N ATOM 3083 CA PRO A 288 −27.682 13.074 18.227 1.00 68.99 C ATOM 3084 C PRO A 288 −27.767 11.903 17.248 1.00 68.90 C ATOM 3085 O PRO A 288 −27.052 10.906 17.406 1.00 67.39 O ATOM 3086 CB PRO A 288 −28.979 13.140 19.057 1.00 68.33 C ATOM 3087 CG PRO A 288 −29.207 14.546 19.305 1.00 66.28 C ATOM 3088 CD PRO A 288 −28.701 15.269 18.091 1.00 70.69 C ATOM 3089 N PHE A 289 −28.641 12.042 16.255 1.00 67.58 N ATOM 3090 CA PHE A 289 −28.807 11.038 15.217 1.00 67.91 C ATOM 3091 C PHE A 289 −27.479 10.746 14.519 1.00 69.22 C ATOM 3092 O PHE A 289 −27.072 9.576 14.412 1.00 68.30 O ATOM 3093 CB PHE A 289 −29.853 11.505 14.189 1.00 69.82 C ATOM 3094 CG PHE A 289 −30.014 10.578 13.047 1.00 67.38 C ATOM 3095 CD1 PHE A 289 −30.850 9.475 13.157 1.00 64.32 C ATOM 3096 CD2 PHE A 289 −29.335 10.795 11.859 1.00 71.12 C ATOM 3097 CE1 PHE A 289 −31.008 8.610 12.114 1.00 70.14 C ATOM 3098 CE2 PHE A 289 −29.489 9.926 10.798 1.00 76.34 C ATOM 3099 CZ PHE A 289 −30.327 8.829 10.923 1.00 75.79 C ATOM 3100 N PHE A 290 −26.818 11.811 14.052 1.00 69.03 N ATOM 3101 CA PHE A 290 −25.522 11.686 13.347 1.00 70.29 C ATOM 3102 C PHE A 290 −24.346 11.254 14.222 1.00 70.41 C ATOM 3103 O PHE A 290 −23.386 10.677 13.697 1.00 72.74 O ATOM 3104 CB PHE A 290 −25.180 12.971 12.585 1.00 73.06 C ATOM 3105 CG PHE A 290 −25.942 13.108 11.295 1.00 73.80 C ATOM 3106 CD1 PHE A 290 −27.103 13.864 11.226 1.00 70.64 C ATOM 3107 CD2 PHE A 290 −25.494 12.467 10.149 1.00 76.58 C ATOM 3108 CE1 PHE A 290 −27.800 13.990 10.058 1.00 79.74 C ATOM 3109 CE2 PHE A 290 −26.191 12.586 8.960 1.00 75.30 C ATOM 3110 CZ PHE A 290 −27.349 13.352 8.914 1.00 77.36 C ATOM 3111 N ILE A 291 −24.404 11.522 15.528 1.00 69.93 N ATOM 3112 CA ILE A 291 −23.391 11.003 16.454 1.00 71.11 C ATOM 3113 C ILE A 291 −23.517 9.476 16.516 1.00 72.55 C ATOM 3114 O ILE A 291 −22.507 8.765 16.534 1.00 76.20 O ATOM 3115 CB ILE A 291 −23.511 11.621 17.873 1.00 71.52 C ATOM 3116 CG1 ILE A 291 −23.073 13.081 17.853 1.00 73.15 C ATOM 3117 CG2 ILE A 291 −22.642 10.888 18.894 1.00 65.02 C ATOM 3118 CD1 ILE A 291 −23.309 13.807 19.140 1.00 72.16 C ATOM 3119 N VAL A 292 −24.757 8.984 16.545 1.00 74.02 N ATOM 3120 CA VAL A 292 −25.023 7.539 16.619 1.00 74.95 C ATOM 3121 C VAL A 292 −24.586 6.782 15.340 1.00 73.29 C ATOM 3122 O VAL A 292 −24.192 5.619 15.426 1.00 74.16 O ATOM 3123 CB VAL A 292 −26.505 7.253 16.998 1.00 74.89 C ATOM 3124 CG1 VAL A 292 −26.814 5.775 16.915 1.00 73.02 C ATOM 3125 CG2 VAL A 292 −26.781 7.774 18.427 1.00 73.75 C ATOM 3126 N ASN A 293 −24.651 7.431 14.176 1.00 73.79 N ATOM 3127 CA ASN A 293 −24.112 6.835 12.936 1.00 72.79 C ATOM 3128 C ASN A 293 −22.641 6.495 13.085 1.00 71.66 C ATOM 3129 O ASN A 293 −22.206 5.409 12.709 1.00 72.11 O ATOM 3130 CB ASN A 293 −24.250 7.782 11.745 1.00 72.08 C ATOM 3131 CG ASN A 293 −25.673 7.961 11.287 1.00 76.70 C ATOM 3132 OD1 ASN A 293 −26.584 7.306 11.777 1.00 77.56 O ATOM 3133 ND2 ASN A 293 −25.871 8.863 10.327 1.00 72.62 N ATOM 3134 N ILE A 294 −21.887 7.435 13.639 1.00 70.24 N ATOM 3135 CA ILE A 294 −20.451 7.272 13.833 1.00 71.74 C ATOM 3136 C ILE A 294 −20.156 6.296 14.963 1.00 71.03 C ATOM 3137 O ILE A 294 −19.288 5.437 14.807 1.00 69.26 O ATOM 3138 CB ILE A 294 −19.784 8.608 14.115 1.00 71.93 C ATOM 3139 CG1 ILE A 294 −19.878 9.502 12.885 1.00 71.27 C ATOM 3140 CG2 ILE A 294 −18.313 8.421 14.498 1.00 72.08 C ATOM 3141 CD1 ILE A 294 −19.391 10.862 13.168 1.00 79.13 C ATOM 3142 N VAL A 295 −20.872 6.426 16.088 1.00 71.55 N ATOM 3143 CA VAL A 295 −20.735 5.484 17.234 1.00 70.36 C ATOM 3144 C VAL A 295 −20.932 4.029 16.782 1.00 69.42 C ATOM 3145 O VAL A 295 −20.224 3.142 17.243 1.00 66.44 O ATOM 3146 CB VAL A 295 −21.720 5.835 18.396 1.00 70.22 C ATOM 3147 CG1 VAL A 295 −21.803 4.721 19.419 1.00 68.33 C ATOM 3148 CG2 VAL A 295 −21.304 7.125 19.065 1.00 71.01 C ATOM 3149 N HIS A 296 −21.890 3.805 15.878 1.00 69.19 N ATOM 3150 CA HIS A 296 −22.128 2.462 15.311 1.00 71.67 C ATOM 3151 C HIS A 296 −21.079 1.977 14.297 1.00 72.30 C ATOM 3152 O HIS A 296 −21.022 0.785 13.996 1.00 72.47 O ATOM 3153 CB HIS A 296 −23.545 2.349 14.737 1.00 72.70 C ATOM 3154 CG HIS A 296 −24.591 2.269 15.795 1.00 77.53 C ATOM 3155 ND1 HIS A 296 −24.369 1.632 16.991 1.00 78.84 N ATOM 3156 CD2 HIS A 296 −25.859 2.736 15.848 1.00 85.05 C ATOM 3157 CE1 HIS A 296 −25.448 1.710 17.733 1.00 78.27 C ATOM 3158 NE2 HIS A 296 −26.370 2.373 17.069 1.00 81.35 N ATOM 3159 N VAL A 297 −20.267 2.893 13.781 1.00 73.43 N ATOM 3160 CA VAL A 297 −19.118 2.535 12.948 1.00 73.61 C ATOM 3161 C VAL A 297 −17.993 2.051 13.878 1.00 72.62 C ATOM 3162 O VAL A 297 −17.220 1.165 13.505 1.00 73.05 O ATOM 3163 CB VAL A 297 −18.653 3.713 12.050 1.00 73.85 C ATOM 3164 CG1 VAL A 297 −17.424 3.338 11.238 1.00 72.56 C ATOM 3165 CG2 VAL A 297 −19.786 4.139 11.127 1.00 72.80 C ATOM 3166 N ILE A 298 −17.912 2.636 15.082 1.00 72.35 N ATOM 3167 CA ILE A 298 −16.930 2.236 16.097 1.00 73.03 C ATOM 3168 C ILE A 298 −17.351 0.886 16.688 1.00 73.53 C ATOM 3169 O ILE A 298 −16.612 −0.099 16.597 1.00 74.08 O ATOM 3170 CB ILE A 298 −16.780 3.288 17.246 1.00 73.65 C ATOM 3171 CG1 ILE A 298 −16.402 4.687 16.716 1.00 76.60 C ATOM 3172 CG2 ILE A 298 −15.757 2.827 18.255 1.00 73.25 C ATOM 3173 CD1 ILE A 298 −15.059 4.764 16.007 1.00 80.21 C ATOM 3174 N GLN A 299 −18.542 0.864 17.290 1.00 73.48 N ATOM 3175 CA GLN A 299 −19.132 −0.345 17.862 1.00 74.55 C ATOM 3176 C GLN A 299 −20.619 −0.354 17.524 1.00 75.17 C ATOM 3177 O GLN A 299 −21.368 0.466 18.040 1.00 77.55 O ATOM 3178 CB GLN A 299 −18.917 −0.401 19.378 1.00 75.38 C ATOM 3179 CG GLN A 299 −19.532 −1.630 20.086 1.00 76.95 C ATOM 3180 CD GLN A 299 −18.993 −2.963 19.563 1.00 80.36 C ATOM 3181 OE1 GLN A 299 −17.801 −3.255 19.675 1.00 81.59 O ATOM 3182 NE2 GLN A 299 −19.883 −3.778 18.992 1.00 79.18 N ATOM 3183 N ASP A 300 −21.035 −1.282 16.662 1.00 75.16 N ATOM 3184 CA ASP A 300 −22.420 −1.357 16.206 1.00 76.26 C ATOM 3185 C ASP A 300 −23.306 −1.994 17.284 1.00 76.70 C ATOM 3186 O ASP A 300 −22.818 −2.756 18.121 1.00 75.98 O ATOM 3187 CB ASP A 300 −22.492 −2.145 14.883 1.00 76.84 C ATOM 3188 CG ASP A 300 −23.781 −1.873 14.069 1.00 78.37 C ATOM 3189 OD1 ASP A 300 −24.504 −0.874 14.303 1.00 80.03 O ATOM 3190 OD2 ASP A 300 −24.068 −2.688 13.168 1.00 83.02 O ATOM 3191 N ASN A 301 −24.601 −1.661 17.248 1.00 76.86 N ATOM 3192 CA ASN A 301 −25.633 −2.196 18.177 1.00 76.17 C ATOM 3193 C ASN A 301 −25.536 −1.740 19.656 1.00 75.65 C ATOM 3194 O ASN A 301 −26.220 −2.290 20.520 1.00 76.59 O ATOM 3195 CB ASN A 301 −25.700 −3.741 18.089 1.00 75.63 C ATOM 3196 CG ASN A 301 −26.022 −4.235 16.690 1.00 74.71 C ATOM 3197 OD1 ASN A 301 −26.917 −3.714 16.030 1.00 64.90 O ATOM 3198 ND2 ASN A 301 −25.291 −5.246 16.233 1.00 75.78 N ATOM 3199 N LEU A 302 −24.700 −0.743 19.938 1.00 76.35 N ATOM 3200 CA LEU A 302 −24.547 −0.197 21.298 1.00 78.80 C ATOM 3201 C LEU A 302 −25.815 0.534 21.817 1.00 79.26 C ATOM 3202 O LEU A 302 −26.074 0.566 23.018 1.00 80.59 O ATOM 3203 CB LEU A 302 −23.350 0.764 21.335 1.00 79.16 C ATOM 3204 CG LEU A 302 −22.832 1.213 22.703 1.00 79.37 C ATOM 3205 CD1 LEU A 302 −22.199 0.039 23.438 1.00 77.36 C ATOM 3206 CD2 LEU A 302 −21.839 2.351 22.545 1.00 79.97 C ATOM 3207 N ILE A 303 −26.572 1.105 20.888 1.00 79.64 N ATOM 3208 CA ILE A 303 −27.765 1.886 21.120 1.00 80.47 C ATOM 3209 C ILE A 303 −28.881 1.166 20.393 1.00 80.12 C ATOM 3210 O ILE A 303 −28.747 0.854 19.208 1.00 80.56 O ATOM 3211 CB ILE A 303 −27.623 3.313 20.573 1.00 79.71 C ATOM 3212 CG1 ILE A 303 −26.513 4.056 21.326 1.00 80.55 C ATOM 3213 CG2 ILE A 303 −28.930 4.068 20.708 1.00 78.77 C ATOM 3214 CD1 ILE A 303 −26.243 5.427 20.826 1.00 83.25 C ATOM 3215 N ARG A 304 −29.973 0.910 21.111 1.00 81.10 N ATOM 3216 CA ARG A 304 −31.132 0.177 20.596 1.00 83.66 C ATOM 3217 C ARG A 304 −31.686 0.776 19.310 1.00 82.48 C ATOM 3218 O ARG A 304 −31.542 1.977 19.070 1.00 83.57 O ATOM 3219 CB ARG A 304 −32.265 0.192 21.608 1.00 84.33 C ATOM 3220 CG ARG A 304 −31.969 −0.426 22.966 1.00 91.17 C ATOM 3221 CD ARG A 304 −33.142 −0.227 23.870 1.00 96.90 C ATOM 3222 NE ARG A 304 −34.321 −0.914 23.347 1.00 107.82 N ATOM 3223 CZ ARG A 304 −35.556 −0.820 23.845 1.00 114.88 C ATOM 3224 NH1 ARG A 304 −35.826 −0.056 24.909 1.00 119.98 N ATOM 3225 NH2 ARG A 304 −36.539 −1.505 23.268 1.00 117.19 N ATOM 3226 N LYS A 305 −32.317 −0.069 18.496 1.00 80.96 N ATOM 3227 CA LYS A 305 −32.950 0.369 17.249 1.00 82.83 C ATOM 3228 C LYS A 305 −34.024 1.432 17.501 1.00 82.19 C ATOM 3229 O LYS A 305 −34.122 2.399 16.751 1.00 83.88 O ATOM 3230 CB LYS A 305 −33.573 −0.819 16.514 1.00 82.92 C ATOM 3231 CG LYS A 305 −34.091 −0.499 15.109 1.00 86.35 C ATOM 3232 CD LYS A 305 −34.754 −1.709 14.472 1.00 87.13 C ATOM 3233 CE LYS A 305 −35.417 −1.329 13.154 1.00 88.05 C ATOM 3234 NZ LYS A 305 −36.147 −2.481 12.552 1.00 91.16 N ATOM 3235 N GLU A 306 −34.813 1.237 18.553 1.00 79.77 N ATOM 3236 CA GLU A 306 −35.888 2.166 18.928 1.00 78.91 C ATOM 3237 C GLU A 306 −35.388 3.534 19.354 1.00 75.42 C ATOM 3238 O GLU A 306 −36.083 4.526 19.156 1.00 73.51 O ATOM 3239 CB GLU A 306 −36.707 1.586 20.062 1.00 80.27 C ATOM 3240 CG GLU A 306 −37.464 0.323 19.682 1.00 88.15 C ATOM 3241 CD GLU A 306 −38.035 −0.379 20.876 1.00 92.42 C ATOM 3242 OE1 GLU A 306 −38.087 0.245 21.964 1.00 103.55 O ATOM 3243 OE2 GLU A 306 −38.436 −1.560 20.740 1.00 104.10 O ATOM 3244 N VAL A 307 −34.191 3.586 19.938 1.00 74.31 N ATOM 3245 CA VAL A 307 −33.579 4.864 20.314 1.00 73.09 C ATOM 3246 C VAL A 307 −33.126 5.563 19.040 1.00 72.26 C ATOM 3247 O VAL A 307 −33.415 6.740 18.854 1.00 74.87 O ATOM 3248 CB VAL A 307 −32.398 4.703 21.330 1.00 71.64 C ATOM 3249 CG1 VAL A 307 −31.597 5.986 21.441 1.00 63.03 C ATOM 3250 CG2 VAL A 307 −32.925 4.287 22.703 1.00 68.24 C ATOM 3251 N TYR A 308 −32.423 4.833 18.177 1.00 71.72 N ATOM 3252 CA TYR A 308 −31.955 5.366 16.892 1.00 72.58 C ATOM 3253 C TYR A 308 −33.106 5.892 16.025 1.00 72.20 C ATOM 3254 O TYR A 308 −32.981 6.951 15.414 1.00 72.72 O ATOM 3255 CB TYR A 308 −31.184 4.294 16.135 1.00 75.16 C ATOM 3256 CG TYR A 308 −30.534 4.770 14.855 1.00 75.61 C ATOM 3257 CD1 TYR A 308 −30.984 4.338 13.610 1.00 83.25 C ATOM 3258 CD2 TYR A 308 −29.470 5.650 14.894 1.00 77.87 C ATOM 3259 CE1 TYR A 308 −30.378 4.780 12.435 1.00 82.87 C ATOM 3260 CE2 TYR A 308 −28.859 6.098 13.747 1.00 74.78 C ATOM 3261 CZ TYR A 308 −29.313 5.663 12.513 1.00 80.83 C ATOM 3262 OH TYR A 308 −28.716 6.100 11.357 1.00 79.13 O ATOM 3263 N ILE A 309 −34.214 5.152 15.979 1.00 70.18 N ATOM 3264 CA ILE A 309 −35.412 5.593 15.251 1.00 71.74 C ATOM 3265 C ILE A 309 −35.959 6.873 15.863 1.00 72.46 C ATOM 3266 O ILE A 309 −36.272 7.798 15.139 1.00 73.87 O ATOM 3267 CB ILE A 309 −36.519 4.520 15.243 1.00 70.27 C ATOM 3268 CG1 ILE A 309 −36.141 3.375 14.316 1.00 69.96 C ATOM 3269 CG2 ILE A 309 −37.839 5.100 14.781 1.00 68.79 C ATOM 3270 CD1 ILE A 309 −36.987 2.145 14.516 1.00 72.32 C ATOM 3271 N LEU A 310 −36.067 6.917 17.195 1.00 74.30 N ATOM 3272 CA LEU A 310 −36.561 8.109 17.895 1.00 73.71 C ATOM 3273 C LEU A 310 −35.682 9.324 17.619 1.00 75.46 C ATOM 3274 O LEU A 310 −36.208 10.416 17.414 1.00 76.09 O ATOM 3275 CB LEU A 310 −36.677 7.873 19.405 1.00 76.16 C ATOM 3276 CG LEU A 310 −37.103 9.087 20.269 1.00 79.17 C ATOM 3277 CD1 LEU A 310 −38.353 9.787 19.699 1.00 80.03 C ATOM 3278 CD2 LEU A 310 −37.344 8.666 21.710 1.00 74.92 C ATOM 3279 N LEU A 311 −34.358 9.142 17.612 1.00 74.63 N ATOM 3280 CA LEU A 311 −33.436 10.249 17.271 1.00 73.90 C ATOM 3281 C LEU A 311 −33.611 10.744 15.829 1.00 73.58 C ATOM 3282 O LEU A 311 −33.346 11.904 15.546 1.00 75.24 O ATOM 3283 CB LEU A 311 −31.976 9.857 17.497 1.00 71.95 C ATOM 3284 CG LEU A 311 −31.583 9.468 18.926 1.00 74.42 C ATOM 3285 CD1 LEU A 311 −30.094 9.202 18.969 1.00 78.05 C ATOM 3286 CD2 LEU A 311 −31.960 10.525 19.951 1.00 79.68 C ATOM 3287 N ASN A 312 −34.053 9.862 14.932 1.00 73.28 N ATOM 3288 CA ASN A 312 −34.337 10.228 13.551 1.00 74.59 C ATOM 3289 C ASN A 312 −35.602 11.076 13.491 1.00 74.96 C ATOM 3290 O ASN A 312 −35.654 12.057 12.741 1.00 76.71 O ATOM 3291 CB ASN A 312 −34.490 8.970 12.679 1.00 73.89 C ATOM 3292 CG ASN A 312 −34.276 9.231 11.170 1.00 75.40 C ATOM 3293 OD1 ASN A 312 −33.933 8.302 10.439 1.00 75.69 O ATOM 3294 ND2 ASN A 312 −34.471 10.471 10.709 1.00 73.01 N ATOM 3295 N TRP A 313 −36.614 10.702 14.275 1.00 75.65 N ATOM 3296 CA TRP A 313 −37.880 11.461 14.327 1.00 73.90 C ATOM 3297 C TRP A 313 −37.748 12.844 14.967 1.00 74.02 C ATOM 3298 O TRP A 313 −38.556 13.726 14.676 1.00 74.92 O ATOM 3299 CB TRP A 313 −38.988 10.634 14.986 1.00 75.64 C ATOM 3300 CG TRP A 313 −39.562 9.768 13.984 1.00 77.34 C ATOM 3301 CD1 TRP A 313 −39.070 8.583 13.548 1.00 77.95 C ATOM 3302 CD2 TRP A 313 −40.757 10.004 13.254 1.00 77.66 C ATOM 3303 NE1 TRP A 313 −39.885 8.056 12.589 1.00 77.47 N ATOM 3304 CE2 TRP A 313 −40.935 8.910 12.384 1.00 79.44 C ATOM 3305 CE3 TRP A 313 −41.701 11.037 13.247 1.00 76.72 C ATOM 3306 CZ2 TRP A 313 −42.025 8.813 11.509 1.00 79.40 C ATOM 3307 CZ3 TRP A 313 −42.790 10.944 12.374 1.00 78.33 C ATOM 3308 CH2 TRP A 313 −42.940 9.835 11.518 1.00 77.48 C ATOM 3309 N ILE A 314 −36.742 13.024 15.822 1.00 71.91 N ATOM 3310 CA ILE A 314 −36.411 14.336 16.368 1.00 71.31 C ATOM 3311 C ILE A 314 −35.847 15.185 15.213 1.00 71.78 C ATOM 3312 O ILE A 314 −36.108 16.396 15.114 1.00 72.42 O ATOM 3313 CB ILE A 314 −35.406 14.239 17.546 1.00 69.93 C ATOM 3314 CG1 ILE A 314 −36.040 13.519 18.735 1.00 69.22 C ATOM 3315 CG2 ILE A 314 −34.975 15.611 18.002 1.00 66.77 C ATOM 3316 CD1 ILE A 314 −35.054 13.195 19.841 1.00 70.69 C ATOM 3317 N GLY A 315 −35.072 14.536 14.350 1.00 71.45 N ATOM 3318 CA GLY A 315 −34.603 15.150 13.118 1.00 73.83 C ATOM 3319 C GLY A 315 −35.780 15.520 12.237 1.00 73.35 C ATOM 3320 O GLY A 315 −35.913 16.673 11.866 1.00 71.64 O ATOM 3321 N TYR A 316 −36.634 14.545 11.909 1.00 74.36 N ATOM 3322 CA TYR A 316 −37.825 14.800 11.050 1.00 74.23 C ATOM 3323 C TYR A 316 −38.668 15.970 11.526 1.00 75.48 C ATOM 3324 O TYR A 316 −38.949 16.889 10.754 1.00 76.19 O ATOM 3325 CB TYR A 316 −38.746 13.570 10.960 1.00 74.65 C ATOM 3326 CG TYR A 316 −38.256 12.359 10.139 1.00 77.26 C ATOM 3327 CD1 TYR A 316 −38.869 11.121 10.288 1.00 77.77 C ATOM 3328 CD2 TYR A 316 −37.200 12.448 9.231 1.00 75.43 C ATOM 3329 CE1 TYR A 316 −38.459 10.025 9.571 1.00 71.53 C ATOM 3330 CE2 TYR A 316 −36.787 11.358 8.514 1.00 78.76 C ATOM 3331 CZ TYR A 316 −37.420 10.148 8.688 1.00 77.38 C ATOM 3332 OH TYR A 316 −37.015 9.061 7.979 1.00 77.21 O ATOM 3333 N VAL A 317 −39.057 15.910 12.801 1.00 74.20 N ATOM 3334 CA VAL A 317 −39.872 16.934 13.466 1.00 72.51 C ATOM 3335 C VAL A 317 −39.316 18.355 13.330 1.00 72.64 C ATOM 3336 O VAL A 317 −40.086 19.311 13.253 1.00 72.17 O ATOM 3337 CB VAL A 317 −40.088 16.560 14.984 1.00 72.14 C ATOM 3338 CG1 VAL A 317 −40.336 17.760 15.804 1.00 74.78 C ATOM 3339 CG2 VAL A 317 −41.253 15.603 15.111 1.00 69.59 C ATOM 3340 N ASN A 318 −37.988 18.491 13.296 1.00 74.61 N ATOM 3341 CA ASN A 318 −37.342 19.793 13.078 1.00 75.44 C ATOM 3342 C ASN A 318 −37.882 20.548 11.857 1.00 75.25 C ATOM 3343 O ASN A 318 −37.912 21.779 11.844 1.00 72.63 O ATOM 3344 CB ASN A 318 −35.843 19.645 12.910 1.00 74.36 C ATOM 3345 CG ASN A 318 −35.171 20.958 12.635 1.00 79.05 C ATOM 3346 OD1 ASN A 318 −35.023 21.806 13.522 1.00 80.80 O ATOM 3347 ND2 ASN A 318 −34.761 21.142 11.396 1.00 83.57 N ATOM 3348 N SER A 319 −38.308 19.798 10.836 1.00 75.32 N ATOM 3349 CA SER A 319 −38.951 20.365 9.640 1.00 76.04 C ATOM 3350 C SER A 319 −40.218 21.194 9.925 1.00 76.24 C ATOM 3351 O SER A 319 −40.670 21.930 9.046 1.00 77.32 O ATOM 3352 CB SER A 319 −39.285 19.245 8.635 1.00 74.82 C ATOM 3353 OG SER A 319 −38.120 18.509 8.281 1.00 77.99 O ATOM 3354 N GLY A 320 −40.782 21.078 11.133 1.00 75.25 N ATOM 3355 CA GLY A 320 −41.934 21.857 11.544 1.00 74.43 C ATOM 3356 C GLY A 320 −41.642 23.110 12.350 1.00 76.72 C ATOM 3357 O GLY A 320 −42.562 23.884 12.581 1.00 78.81 O ATOM 3358 N PHE A 321 −40.391 23.328 12.777 1.00 75.79 N ATOM 3359 CA PHE A 321 −40.076 24.440 13.706 1.00 77.01 C ATOM 3360 C PHE A 321 −39.761 25.780 13.112 1.00 77.35 C ATOM 3361 O PHE A 321 −40.178 26.799 13.680 1.00 75.78 O ATOM 3362 CB PHE A 321 −38.912 24.051 14.634 1.00 78.53 C ATOM 3363 CG PHE A 321 −39.192 22.851 15.497 1.00 78.83 C ATOM 3364 CD1 PHE A 321 −38.154 22.005 15.857 1.00 82.01 C ATOM 3365 CD2 PHE A 321 −40.490 22.551 15.953 1.00 81.40 C ATOM 3366 CE1 PHE A 321 −38.383 20.891 16.650 1.00 81.76 C ATOM 3367 CE2 PHE A 321 −40.722 21.446 16.740 1.00 83.47 C ATOM 3368 CZ PHE A 321 −39.667 20.614 17.091 1.00 85.30 C ATOM 3369 N ASN A 322 −39.040 25.807 11.996 1.00 77.86 N ATOM 3370 CA ASN A 322 −38.637 27.082 11.381 1.00 78.02 C ATOM 3371 C ASN A 322 −39.785 28.061 11.142 1.00 78.43 C ATOM 3372 O ASN A 322 −39.620 29.237 11.440 1.00 77.20 O ATOM 3373 CB ASN A 322 −37.866 26.856 10.087 1.00 79.19 C ATOM 3374 CG ASN A 322 −36.531 26.208 10.315 1.00 81.79 C ATOM 3375 OD1 ASN A 322 −36.158 25.901 11.455 1.00 74.61 O ATOM 3376 ND2 ASN A 322 −35.791 25.989 9.229 1.00 85.04 N ATOM 3377 N PRO A 323 −40.941 27.582 10.609 1.00 78.55 N ATOM 3378 CA PRO A 323 −42.088 28.479 10.469 1.00 79.23 C ATOM 3379 C PRO A 323 −42.469 29.179 11.779 1.00 80.55 C ATOM 3380 O PRO A 323 −42.795 30.367 11.751 1.00 83.61 O ATOM 3381 CB PRO A 323 −43.200 27.542 9.997 1.00 80.04 C ATOM 3382 CG PRO A 323 −42.474 26.473 9.266 1.00 79.60 C ATOM 3383 CD PRO A 323 −41.271 26.232 10.104 1.00 78.83 C ATOM 3384 N LEU A 324 −42.426 28.454 12.899 1.00 80.72 N ATOM 3385 CA LEU A 324 −42.689 29.050 14.233 1.00 81.22 C ATOM 3386 C LEU A 324 −41.580 30.013 14.651 1.00 78.60 C ATOM 3387 O LEU A 324 −41.851 31.053 15.230 1.00 77.91 O ATOM 3388 CB LEU A 324 −42.847 27.970 15.310 1.00 83.48 C ATOM 3389 CG LEU A 324 −44.017 27.003 15.125 1.00 90.04 C ATOM 3390 CD1 LEU A 324 −43.821 25.727 15.948 1.00 91.90 C ATOM 3391 CD2 LEU A 324 −45.329 27.707 15.474 1.00 93.47 C ATOM 3392 N ILE A 325 −40.334 29.656 14.355 1.00 77.22 N ATOM 3393 CA ILE A 325 −39.186 30.504 14.689 1.00 76.61 C ATOM 3394 C ILE A 325 −39.244 31.830 13.907 1.00 75.96 C ATOM 3395 O ILE A 325 −38.791 32.850 14.407 1.00 76.16 O ATOM 3396 CB ILE A 325 −37.837 29.776 14.433 1.00 76.87 C ATOM 3397 CG1 ILE A 325 −37.701 28.520 15.328 1.00 77.05 C ATOM 3398 CG2 ILE A 325 −36.654 30.701 14.689 1.00 73.66 C ATOM 3399 CD1 ILE A 325 −36.611 27.536 14.870 1.00 70.46 C ATOM 3400 N TYR A 326 −39.803 31.815 12.690 1.00 76.28 N ATOM 3401 CA TYR A 326 −39.934 33.054 11.884 1.00 76.44 C ATOM 3402 C TYR A 326 −40.978 34.017 12.416 1.00 73.86 C ATOM 3403 O TYR A 326 −40.991 35.153 11.992 1.00 77.86 O ATOM 3404 CB TYR A 326 −40.242 32.772 10.409 1.00 77.72 C ATOM 3405 CG TYR A 326 −39.240 31.893 9.701 1.00 77.26 C ATOM 3406 CD1 TYR A 326 −39.666 30.889 8.847 1.00 77.44 C ATOM 3407 CD2 TYR A 326 −37.865 32.057 9.885 1.00 81.33 C ATOM 3408 CE1 TYR A 326 −38.764 30.077 8.194 1.00 78.66 C ATOM 3409 CE2 TYR A 326 −36.961 31.248 9.236 1.00 83.58 C ATOM 3410 CZ TYR A 326 −37.423 30.258 8.389 1.00 80.70 C ATOM 3411 OH TYR A 326 −36.541 29.451 7.742 1.00 81.84 O ATOM 3412 N CYS A 327 −41.848 33.582 13.332 1.00 72.88 N ATOM 3413 CA CYS A 327 −42.782 34.496 13.999 1.00 76.25 C ATOM 3414 C CYS A 327 −42.064 35.537 14.905 1.00 78.27 C ATOM 3415 O CYS A 327 −42.684 36.508 15.330 1.00 79.91 O ATOM 3416 CB CYS A 327 −43.827 33.729 14.805 1.00 77.89 C ATOM 3417 SG CYS A 327 −44.970 32.742 13.778 1.00 82.26 S ATOM 3418 N ARG A 328 −40.770 35.320 15.191 1.00 79.14 N ATOM 3419 CA ARG A 328 −39.919 36.324 15.832 1.00 79.59 C ATOM 3420 C ARG A 328 −39.781 37.596 14.984 1.00 79.99 C ATOM 3421 O ARG A 328 −39.581 38.680 15.524 1.00 79.93 O ATOM 3422 CB ARG A 328 −38.515 35.771 16.078 1.00 77.25 C ATOM 3423 CG ARG A 328 −38.423 34.646 17.130 1.00 76.09 C ATOM 3424 CD ARG A 328 −36.998 34.067 17.220 1.00 77.31 C ATOM 3425 NE ARG A 328 −36.006 35.129 17.048 1.00 75.58 N ATOM 3426 CZ ARG A 328 −35.537 35.945 17.985 1.00 81.23 C ATOM 3427 NH1 ARG A 328 −35.953 35.859 19.252 1.00 74.46 N ATOM 3428 NH2 ARG A 328 −34.631 36.866 17.640 1.00 81.80 N ATOM 3429 N SER A 329 −39.885 37.449 13.659 1.00 82.57 N ATOM 3430 CA SER A 329 −39.788 38.574 12.743 1.00 84.20 C ATOM 3431 C SER A 329 −41.107 39.324 12.711 1.00 84.79 C ATOM 3432 O SER A 329 −42.151 38.687 12.597 1.00 88.37 O ATOM 3433 CB SER A 329 −39.460 38.097 11.335 1.00 84.66 C ATOM 3434 OG SER A 329 −39.465 39.190 10.429 1.00 90.44 O ATOM 3435 N PRO A 330 −41.076 40.675 12.814 1.00 86.62 N ATOM 3436 CA PRO A 330 −42.339 41.415 12.685 1.00 85.84 C ATOM 3437 C PRO A 330 −42.905 41.369 11.253 1.00 86.45 C ATOM 3438 O PRO A 330 −44.103 41.466 11.079 1.00 85.67 O ATOM 3439 CB PRO A 330 −41.960 42.851 13.080 1.00 85.22 C ATOM 3440 CG PRO A 330 −40.597 42.764 13.669 1.00 85.83 C ATOM 3441 CD PRO A 330 −39.946 41.595 13.045 1.00 85.56 C ATOM 3442 N ASP A 331 −42.031 41.216 10.256 1.00 87.75 N ATOM 3443 CA ASP A 331 −42.440 41.127 8.854 1.00 88.35 C ATOM 3444 C ASP A 331 −43.160 39.831 8.516 1.00 88.54 C ATOM 3445 O ASP A 331 −44.180 39.865 7.824 1.00 87.13 O ATOM 3446 CB ASP A 331 −41.225 41.268 7.929 1.00 91.75 C ATOM 3447 CG ASP A 331 −40.585 42.640 8.000 1.00 99.65 C ATOM 3448 OD1 ASP A 331 −41.152 43.526 8.660 1.00 101.57 O ATOM 3449 OD2 ASP A 331 −39.507 42.825 7.387 1.00 108.72 O ATOM 3450 N PHE A 332 −42.638 38.697 8.992 1.00 87.91 N ATOM 3451 CA PHE A 332 −43.306 37.387 8.759 1.00 85.83 C ATOM 3452 C PHE A 332 −44.596 37.298 9.525 1.00 84.97 C ATOM 3453 O PHE A 332 −45.583 36.811 9.006 1.00 85.85 O ATOM 3454 CB PHE A 332 −42.419 36.206 9.142 1.00 83.02 C ATOM 3455 CG PHE A 332 −41.421 35.848 8.099 1.00 81.58 C ATOM 3456 CD1 PHE A 332 −40.184 36.470 8.064 1.00 79.60 C ATOM 3457 CD2 PHE A 332 −41.719 34.884 7.141 1.00 82.96 C ATOM 3458 CE1 PHE A 332 −39.251 36.138 7.092 1.00 83.90 C ATOM 3459 CE2 PHE A 332 −40.794 34.541 6.159 1.00 81.77 C ATOM 3460 CZ PHE A 332 −39.558 35.166 6.132 1.00 84.78 C ATOM 3461 N ARG A 333 −44.567 37.772 10.764 1.00 85.64 N ATOM 3462 CA ARG A 333 −45.742 37.795 11.618 1.00 86.23 C ATOM 3463 C ARG A 333 −46.875 38.603 10.964 1.00 85.68 C ATOM 3464 O ARG A 333 −48.015 38.171 11.005 1.00 85.47 O ATOM 3465 CB ARG A 333 −45.357 38.362 12.963 1.00 86.76 C ATOM 3466 CG ARG A 333 −46.383 38.222 14.059 1.00 90.70 C ATOM 3467 CD ARG A 333 −45.671 38.263 15.407 1.00 93.90 C ATOM 3468 NE ARG A 333 −44.710 39.377 15.472 1.00 100.54 N ATOM 3469 CZ ARG A 333 −43.693 39.490 16.335 1.00 103.24 C ATOM 3470 NH1 ARG A 333 −43.449 38.553 17.262 1.00 104.89 N ATOM 3471 NH2 ARG A 333 −42.899 40.566 16.271 1.00 102.64 N ATOM 3472 N ILE A 334 −46.534 39.755 10.373 1.00 84.51 N ATOM 3473 CA ILE A 334 −47.472 40.580 9.584 1.00 83.97 C ATOM 3474 C ILE A 334 −47.898 39.862 8.316 1.00 82.02 C ATOM 3475 O ILE A 334 −49.077 39.871 7.963 1.00 86.53 O ATOM 3476 CB ILE A 334 −46.862 41.959 9.211 1.00 85.26 C ATOM 3477 CG1 ILE A 334 −46.777 42.860 10.444 1.00 85.90 C ATOM 3478 CG2 ILE A 334 −47.692 42.668 8.142 1.00 82.88 C ATOM 3479 CD1 ILE A 334 −45.791 44.015 10.287 1.00 88.19 C ATOM 3480 N ALA A 335 −46.934 39.248 7.635 1.00 80.09 N ATOM 3481 CA ALA A 335 −47.202 38.470 6.433 1.00 79.44 C ATOM 3482 C ALA A 335 −48.199 37.345 6.719 1.00 78.78 C ATOM 3483 O ALA A 335 −49.207 37.251 6.057 1.00 78.15 O ATOM 3484 CB ALA A 335 −45.911 37.896 5.876 1.00 78.92 C ATOM 3485 N PHE A 336 −47.896 36.511 7.714 1.00 78.39 N ATOM 3486 CA PHE A 336 −48.760 35.372 8.092 1.00 77.87 C ATOM 3487 C PHE A 336 −50.184 35.751 8.427 1.00 77.70 C ATOM 3488 O PHE A 336 −51.116 35.205 7.847 1.00 75.76 O ATOM 3489 CB PHE A 336 −48.198 34.602 9.301 1.00 77.26 C ATOM 3490 CG PHE A 336 −46.852 33.943 9.072 1.00 77.36 C ATOM 3491 CD1 PHE A 336 −46.408 33.562 7.796 1.00 78.61 C ATOM 3492 CD2 PHE A 336 −46.024 33.695 10.152 1.00 76.74 C ATOM 3493 CE1 PHE A 336 −45.180 32.962 7.621 1.00 76.21 C ATOM 3494 CE2 PHE A 336 −44.787 33.089 9.974 1.00 78.36 C ATOM 3495 CZ PHE A 336 −44.372 32.724 8.700 1.00 76.78 C ATOM 3496 N GLN A 337 −50.346 36.681 9.363 1.00 78.23 N ATOM 3497 CA GLN A 337 −51.691 37.124 9.792 1.00 79.51 C ATOM 3498 C GLN A 337 −52.549 37.711 8.642 1.00 78.03 C ATOM 3499 O GLN A 337 −53.778 37.646 8.694 1.00 78.24 O ATOM 3500 CB GLN A 337 −51.609 38.081 10.997 1.00 80.74 C ATOM 3501 CG GLN A 337 −50.870 39.391 10.802 1.00 86.86 C ATOM 3502 CD GLN A 337 −50.613 40.113 12.143 1.00 89.20 C ATOM 3503 OE1 GLN A 337 −51.317 39.877 13.127 1.00 97.34 O ATOM 3504 NE2 GLN A 337 −49.610 40.989 12.177 1.00 94.24 N ATOM 3505 N GLU A 338 −51.900 38.271 7.622 1.00 76.38 N ATOM 3506 CA GLU A 338 −52.591 38.694 6.411 1.00 74.47 C ATOM 3507 C GLU A 338 −53.085 37.448 5.670 1.00 73.86 C ATOM 3508 O GLU A 338 −54.258 37.370 5.282 1.00 73.06 O ATOM 3509 CB GLU A 338 −51.659 39.512 5.516 1.00 75.29 C ATOM 3510 CG GLU A 338 −52.343 40.279 4.365 1.00 79.56 C ATOM 3511 CD GLU A 338 −52.695 39.442 3.113 1.00 85.28 C ATOM 3512 OE1 GLU A 338 −52.500 38.207 3.087 1.00 91.20 O ATOM 3513 OE2 GLU A 338 −53.176 40.048 2.131 1.00 89.12 O ATOM 3514 N LEU A 339 −52.177 36.488 5.477 1.00 70.47 N ATOM 3515 CA LEU A 339 −52.475 35.242 4.763 1.00 71.14 C ATOM 3516 C LEU A 339 −53.527 34.375 5.463 1.00 72.95 C ATOM 3517 O LEU A 339 −54.255 33.650 4.793 1.00 74.46 O ATOM 3518 CB LEU A 339 −51.209 34.406 4.567 1.00 68.65 C ATOM 3519 CG LEU A 339 −50.055 35.021 3.778 1.00 69.52 C ATOM 3520 CD1 LEU A 339 −48.805 34.139 3.935 1.00 68.79 C ATOM 3521 CD2 LEU A 339 −50.411 35.225 2.315 1.00 62.62 C ATOM 3522 N LEU A 340 −53.599 34.455 6.796 1.00 74.28 N ATOM 3523 CA LEU A 340 −54.598 33.718 7.588 1.00 75.08 C ATOM 3524 C LEU A 340 −55.848 34.573 7.901 1.00 76.80 C ATOM 3525 O LEU A 340 −56.573 34.280 8.860 1.00 76.39 O ATOM 3526 CB LEU A 340 −53.960 33.174 8.878 1.00 74.68 C ATOM 3527 CG LEU A 340 −52.763 32.233 8.701 1.00 73.84 C ATOM 3528 CD1 LEU A 340 −52.150 31.887 10.048 1.00 73.83 C ATOM 3529 CD2 LEU A 340 −53.167 30.976 7.982 1.00 73.24 C ATOM 3530 N CYS A 341 −56.083 35.619 7.088 1.00 79.59 N ATOM 3531 CA CYS A 341 −57.280 36.477 7.152 1.00 80.75 C ATOM 3532 C CYS A 341 −57.536 37.089 8.530 1.00 81.21 C ATOM 3533 O CYS A 341 −58.613 36.911 9.114 1.00 81.97 O ATOM 3534 CB CYS A 341 −58.500 35.683 6.674 1.00 81.26 C ATOM 3535 SG CYS A 341 −58.243 34.889 5.079 1.00 88.24 S ATOM 3536 N LEU A 342 −56.533 37.809 9.031 1.00 81.84 N ATOM 3537 CA LEU A 342 −56.598 38.483 10.333 1.00 82.60 C ATOM 3538 C LEU A 342 −56.370 39.993 10.158 1.00 83.35 C ATOM 3539 O LEU A 342 −55.715 40.650 10.971 1.00 85.10 O ATOM 3540 CB LEU A 342 −55.575 37.878 11.303 1.00 82.19 C ATOM 3541 CG LEU A 342 −55.513 36.347 11.407 1.00 82.55 C ATOM 3542 CD1 LEU A 342 −54.415 35.913 12.368 1.00 82.95 C ATOM 3543 CD2 LEU A 342 −56.851 35.782 11.838 1.00 83.45 C TER 3544 LEU A 342 HETATM 3545 C1 MAL A 401 −30.433 67.551 22.604 1.00 116.54 C HETATM 3546 C2 MAL A 401 −29.508 68.271 21.613 1.00 116.67 C HETATM 3547 C3 MAL A 401 −28.286 67.466 21.250 1.00 115.67 C HETATM 3548 C4 MAL A 401 −27.437 67.774 22.475 1.00 113.35 C HETATM 3549 C5 MAL A 401 −28.222 67.326 23.742 1.00 114.16 C HETATM 3550 C6 MAL A 401 −27.467 67.821 24.997 1.00 112.25 C HETATM 3551 O1 MAL A 401 −30.860 66.152 22.481 1.00 118.82 O HETATM 3552 O2 MAL A 401 −30.196 68.896 20.430 1.00 113.17 O HETATM 3553 O3 MAL A 401 −27.692 67.923 20.042 1.00 118.88 O HETATM 3554 O4 MAL A 401 −26.151 67.133 22.417 1.00 110.96 O HETATM 3555 O5 MAL A 401 −29.640 67.723 23.796 1.00 116.03 O HETATM 3556 O6 MAL A 401 −28.329 68.191 26.091 1.00 111.18 O HETATM 3557 C1′ MAL A 401 −34.225 64.365 24.555 1.00 118.64 C HETATM 3558 C2′ MAL A 401 −34.062 65.875 24.695 1.00 118.98 C HETATM 3559 C3′ MAL A 401 −33.053 66.497 23.732 1.00 118.89 C HETATM 3560 C4′ MAL A 401 −31.773 65.653 23.533 1.00 118.41 C HETATM 3561 C5′ MAL A 401 −32.192 64.169 23.329 1.00 117.27 C HETATM 3562 C6′ MAL A 401 −31.044 63.164 23.154 1.00 114.49 C HETATM 3563 O1′ MAL A 401 −34.905 63.850 25.705 1.00 115.98 O HETATM 3564 O2′ MAL A 401 −35.328 66.510 24.463 1.00 120.41 O HETATM 3565 O3′ MAL A 401 −32.803 67.771 24.346 1.00 118.98 O HETATM 3566 O5′ MAL A 401 −32.951 63.737 24.467 1.00 118.26 O HETATM 3567 O6′ MAL A 401 −31.319 61.889 23.781 1.00 101.09 O HETATM 3568 S SO4 A 402 −30.549 40.587 1.840 1.00 88.35 S HETATM 3569 O1 SO4 A 402 −31.012 40.657 0.449 1.00 91.49 O HETATM 3570 O2 SO4 A 402 −31.341 41.518 2.648 1.00 79.98 O HETATM 3571 O3 SO4 A 402 −30.747 39.233 2.389 1.00 86.89 O HETATM 3572 O4 SO4 A 402 −29.136 40.942 1.808 1.00 79.16 O HETATM 3573 S SO4 A 403 −34.529 39.154 20.841 1.00 91.42 S HETATM 3574 O1 SO4 A 403 −35.518 40.193 21.131 1.00 84.45 O HETATM 3575 O2 SO4 A 403 −33.296 39.334 21.612 1.00 93.22 O HETATM 3576 O3 SO4 A 403 −35.078 37.875 21.232 1.00 84.98 O HETATM 3577 O4 SO4 A 403 −34.193 39.246 19.438 1.00 95.59 O HETATM 3578 S SO4 A 404 −26.677 61.410 43.667 1.00 79.61 S HETATM 3579 O1 SO4 A 404 −26.224 60.482 42.637 1.00 81.00 O HETATM 3580 O2 SO4 A 404 −27.968 62.001 43.315 1.00 78.07 O HETATM 3581 O3 SO4 A 404 −26.852 60.655 44.907 1.00 85.67 O HETATM 3582 O4 SO4 A 404 −25.700 62.487 43.819 1.00 76.33 O HETATM 3583 S SO4 A 405 −38.222 54.167 28.613 1.00 76.15 S HETATM 3584 O1 SO4 A 405 −36.815 54.454 28.371 1.00 62.35 O HETATM 3585 O2 SO4 A 405 −38.708 53.202 27.628 1.00 74.38 O HETATM 3586 O3 SO4 A 405 −38.392 53.561 29.925 1.00 77.47 O HETATM 3587 O4 SO4 A 405 −38.983 55.412 28.536 1.00 74.20 O HETATM 3588 S SO4 A 406 −14.626 46.742 34.955 1.00 133.97 S HETATM 3589 O1 SO4 A 406 −14.905 46.888 33.527 1.00 132.94 O HETATM 3590 O2 SO4 A 406 −15.870 46.485 35.690 1.00 131.52 O HETATM 3591 O3 SO4 A 406 −13.744 45.588 35.141 1.00 137.48 O HETATM 3592 O4 SO4 A 406 −13.968 47.953 35.460 1.00 129.69 O HETATM 3593 S SO4 A 407 −39.375 59.242 10.957 1.00 103.03 S HETATM 3594 O1 SO4 A 407 −38.112 58.591 10.589 1.00 100.34 O HETATM 3595 O2 SO4 A 407 −40.365 59.029 9.899 1.00 104.55 O HETATM 3596 O3 SO4 A 407 −39.865 58.663 12.211 1.00 107.47 O HETATM 3597 O4 SO4 A 407 −39.174 60.683 11.138 1.00 106.88 O HETATM 3598 O17 CAU A 408 −33.477 10.957 8.170 1.00 50.96 O HETATM 3599 C16 CAU A 408 −32.267 10.230 8.041 1.00 45.65 C HETATM 3600 C18 CAU A 408 −32.478 8.951 7.225 1.00 51.24 C HETATM 3601 N19 CAU A 408 −33.702 8.250 7.600 1.00 54.99 N HETATM 3602 C20 CAU A 408 −33.806 6.805 7.498 1.00 60.13 C HETATM 3603 C21 CAU A 408 −33.533 6.385 6.055 1.00 66.62 C HETATM 3604 C22 CAU A 408 −35.184 6.350 7.988 1.00 59.87 C HETATM 3605 C15 CAU A 408 −31.242 11.105 7.364 1.00 46.24 C HETATM 3606 O14 CAU A 408 −30.049 10.367 7.182 1.00 51.01 O HETATM 3607 C13 CAU A 408 −28.931 10.857 6.581 1.00 52.29 C HETATM 3608 C12 CAU A 408 −28.911 12.133 6.005 1.00 56.44 C HETATM 3609 C11 CAU A 408 −27.768 12.628 5.393 1.00 56.29 C HETATM 3610 C10 CAU A 408 −26.608 11.875 5.332 1.00 54.90 C HETATM 3611 C8 CAU A 408 −26.565 10.625 5.875 1.00 54.68 C HETATM 3612 C9 CAU A 408 −27.768 10.108 6.517 1.00 53.44 C HETATM 3613 N7 CAU A 408 −25.593 9.657 5.974 1.00 57.07 N HETATM 3614 C3 CAU A 408 −26.096 8.561 6.637 1.00 55.09 C HETATM 3615 C4 CAU A 408 −27.482 8.863 6.976 1.00 53.66 C HETATM 3616 C2 CAU A 408 −25.589 7.338 7.009 1.00 54.54 C HETATM 3617 C1 CAU A 408 −26.395 6.432 7.689 1.00 56.87 C HETATM 3618 C6 CAU A 408 −27.717 6.731 8.006 1.00 56.28 C HETATM 3619 C5 CAU A 408 −28.269 7.948 7.652 1.00 56.42 C HETATM 3620 C1 BU1 A 409 −26.142 41.424 19.127 1.00 52.74 C HETATM 3621 C2 BU1 A 409 −24.673 41.194 19.349 1.00 50.50 C HETATM 3622 C3 BU1 A 409 −24.361 39.703 19.256 1.00 58.68 C HETATM 3623 C4 BU1 A 409 −23.639 39.193 20.492 1.00 60.90 C HETATM 3624 O5 BU1 A 409 −26.449 42.812 19.063 1.00 46.89 O HETATM 3625 O6 BU1 A 409 −23.397 37.796 20.358 1.00 65.39 O HETATM 3626 C1 BU1 A 410 −25.492 26.523 17.727 1.00 80.96 C HETATM 3627 C2 BU1 A 410 −25.259 26.818 16.240 1.00 75.39 C HETATM 3628 C3 BU1 A 410 −25.569 28.286 15.904 1.00 72.90 C HETATM 3629 C4 BU1 A 410 −24.368 29.076 15.393 1.00 70.20 C HETATM 3630 O5 BU1 A 410 −25.036 25.202 18.096 1.00 82.37 O HETATM 3631 O6 BU1 A 410 −23.728 29.769 16.468 1.00 72.23 O HETATM 3632 C1 ACM A 411 −33.365 45.116 23.775 1.00 72.09 C HETATM 3633 O ACM A 411 −34.580 45.100 23.784 1.00 74.74 O HETATM 3634 N ACM A 411 −32.677 43.981 23.897 1.00 71.73 N HETATM 3635 C2 ACM A 411 −32.587 46.397 23.626 1.00 69.99 C HETATM 3636 C1 CLR A 412 −36.292 27.653 −4.352 1.00 98.97 C HETATM 3637 C2 CLR A 412 −36.571 29.133 −4.591 1.00 98.10 C HETATM 3638 C3 CLR A 412 −35.425 29.793 −5.354 1.00 98.91 C HETATM 3639 C4 CLR A 412 −34.965 29.038 −6.627 1.00 98.80 C HETATM 3640 C5 CLR A 412 −35.001 27.515 −6.513 1.00 99.38 C HETATM 3641 C6 CLR A 412 −34.088 26.785 −7.209 1.00 99.72 C HETATM 3642 C7 CLR A 412 −34.017 25.259 −7.204 1.00 98.91 C HETATM 3643 C8 CLR A 412 −35.275 24.631 −6.633 1.00 99.40 C HETATM 3644 C9 CLR A 412 −35.651 25.377 −5.337 1.00 99.32 C HETATM 3645 C10 CLR A 412 −36.050 26.846 −5.645 1.00 100.31 C HETATM 3646 C11 CLR A 412 −36.707 24.655 −4.466 1.00 96.50 C HETATM 3647 C12 CLR A 412 −36.536 23.143 −4.365 1.00 96.65 C HETATM 3648 C13 CLR A 412 −36.329 22.495 −5.719 1.00 100.10 C HETATM 3649 C14 CLR A 412 −35.100 23.138 −6.353 1.00 101.09 C HETATM 3650 C15 CLR A 412 −34.782 22.237 −7.542 1.00 101.42 C HETATM 3651 C16 CLR A 412 −35.217 20.837 −7.081 1.00 101.37 C HETATM 3652 C17 CLR A 412 −35.995 21.000 −5.758 1.00 100.60 C HETATM 3653 C18 CLR A 412 −37.562 22.726 −6.571 1.00 103.09 C HETATM 3654 C19 CLR A 412 −37.268 26.936 −6.512 1.00 102.37 C HETATM 3655 C20 CLR A 412 −37.144 19.961 −5.632 1.00 100.25 C HETATM 3656 C21 CLR A 412 −38.032 20.187 −4.406 1.00 98.50 C HETATM 3657 C22 CLR A 412 −36.561 18.538 −5.657 1.00 101.01 C HETATM 3658 C23 CLR A 412 −37.437 17.457 −5.012 1.00 103.22 C HETATM 3659 C24 CLR A 412 −36.748 16.094 −5.099 1.00 105.36 C HETATM 3660 C25 CLR A 412 −37.535 15.014 −5.853 1.00 107.48 C HETATM 3661 C26 CLR A 412 −36.571 14.080 −6.591 1.00 107.73 C HETATM 3662 C27 CLR A 412 −38.402 14.192 −4.899 1.00 106.61 C HETATM 3663 O1 CLR A 412 −35.828 31.158 −5.664 1.00 99.70 O HETATM 3664 C1 CLR A 413 −52.083 29.442 3.198 1.00 84.76 C HETATM 3665 C2 CLR A 413 −52.244 30.951 3.100 1.00 85.75 C HETATM 3666 C3 CLR A 413 −53.214 31.296 1.956 1.00 84.48 C HETATM 3667 C4 CLR A 413 −54.606 30.718 2.233 1.00 82.59 C HETATM 3668 C5 CLR A 413 −54.424 29.218 2.245 1.00 86.97 C HETATM 3669 C6 CLR A 413 −55.171 28.468 1.400 1.00 86.01 C HETATM 3670 C7 CLR A 413 −55.077 26.968 1.309 1.00 88.00 C HETATM 3671 C8 CLR A 413 −54.340 26.362 2.487 1.00 89.33 C HETATM 3672 C9 CLR A 413 −53.087 27.161 2.862 1.00 88.72 C HETATM 3673 C10 CLR A 413 −53.399 28.636 3.214 1.00 85.54 C HETATM 3674 C11 CLR A 413 −52.265 26.440 3.959 1.00 87.79 C HETATM 3675 C12 CLR A 413 −51.961 24.981 3.616 1.00 90.38 C HETATM 3676 C13 CLR A 413 −53.219 24.202 3.283 1.00 90.23 C HETATM 3677 C14 CLR A 413 −53.904 24.933 2.142 1.00 92.32 C HETATM 3678 C15 CLR A 413 −54.976 23.971 1.668 1.00 92.65 C HETATM 3679 C16 CLR A 413 −54.282 22.616 1.827 1.00 93.79 C HETATM 3680 C17 CLR A 413 −53.018 22.820 2.693 1.00 91.53 C HETATM 3681 C18 CLR A 413 −54.156 24.093 4.499 1.00 87.86 C HETATM 3682 C19 CLR A 413 −54.039 28.774 4.595 1.00 82.64 C HETATM 3683 C20 CLR A 413 −52.772 21.624 3.653 1.00 91.51 C HETATM 3684 C21 CLR A 413 −51.815 21.878 4.816 1.00 89.13 C HETATM 3685 C22 CLR A 413 −52.233 20.430 2.857 1.00 91.91 C HETATM 3686 C23 CLR A 413 −52.841 19.099 3.296 1.00 93.30 C HETATM 3687 C24 CLR A 413 −52.459 17.993 2.311 1.00 92.99 C HETATM 3688 C25 CLR A 413 −51.938 16.746 3.002 1.00 92.39 C HETATM 3689 C26 CLR A 413 −52.059 15.541 2.076 1.00 94.36 C HETATM 3690 C27 CLR A 413 −50.499 16.952 3.460 1.00 89.59 C HETATM 3691 O1 CLR A 413 −53.334 32.684 1.607 1.00 81.53 O HETATM 3692 C1 CLR A 414 −41.706 27.953 −4.419 1.00 114.97 C HETATM 3693 C2 CLR A 414 −41.768 29.448 −4.709 1.00 114.73 C HETATM 3694 C3 CLR A 414 −40.664 29.838 −5.694 1.00 114.23 C HETATM 3695 C4 CLR A 414 −40.711 29.022 −7.000 1.00 115.07 C HETATM 3696 C5 CLR A 414 −40.836 27.528 −6.714 1.00 115.29 C HETATM 3697 C6 CLR A 414 −40.069 26.669 −7.418 1.00 115.44 C HETATM 3698 C7 CLR A 414 −40.081 25.169 −7.226 1.00 116.17 C HETATM 3699 C8 CLR A 414 −41.303 24.678 −6.473 1.00 115.82 C HETATM 3700 C9 CLR A 414 −41.567 25.570 −5.252 1.00 114.14 C HETATM 3701 C10 CLR A 414 −41.815 27.052 −5.652 1.00 114.08 C HETATM 3702 C11 CLR A 414 −42.685 24.996 −4.348 1.00 113.90 C HETATM 3703 C12 CLR A 414 −42.459 23.526 −3.999 1.00 115.68 C HETATM 3704 C13 CLR A 414 −42.294 22.667 −5.238 1.00 115.89 C HETATM 3705 C14 CLR A 414 −41.123 23.217 −6.042 1.00 116.05 C HETATM 3706 C15 CLR A 414 −40.935 22.171 −7.143 1.00 117.57 C HETATM 3707 C16 CLR A 414 −41.285 20.849 −6.450 1.00 117.86 C HETATM 3708 C17 CLR A 414 −41.874 21.195 −5.060 1.00 116.61 C HETATM 3709 C18 CLR A 414 −43.602 22.724 −6.051 1.00 114.74 C HETATM 3710 C19 CLR A 414 −43.197 27.247 −6.285 1.00 114.74 C HETATM 3711 C20 CLR A 414 −42.937 20.193 −4.516 1.00 116.12 C HETATM 3712 C21 CLR A 414 −43.284 20.508 −3.065 1.00 114.86 C HETATM 3713 C22 CLR A 414 −42.503 18.733 −4.603 1.00 115.06 C HETATM 3714 C23 CLR A 414 −43.586 17.708 −4.216 1.00 115.00 C HETATM 3715 C24 CLR A 414 −42.985 16.411 −3.662 1.00 113.44 C HETATM 3716 C25 CLR A 414 −42.272 15.571 −4.726 1.00 113.32 C HETATM 3717 C26 CLR A 414 −40.869 16.077 −5.018 1.00 111.52 C HETATM 3718 C27 CLR A 414 −42.204 14.107 −4.309 1.00 112.05 C HETATM 3719 O1 CLR A 414 −40.674 31.263 −5.966 1.00 114.05 O HETATM 3720 C1 PLM A 415 −57.959 33.303 4.949 1.00 94.70 C HETATM 3721 O2 PLM A 415 −57.047 33.732 4.211 1.00 99.49 O HETATM 3722 C2 PLM A 415 −58.012 31.828 5.279 1.00 93.74 C HETATM 3723 C3 PLM A 415 −59.131 31.125 4.492 1.00 92.47 C HETATM 3724 C4 PLM A 415 −58.642 29.982 3.590 1.00 91.37 C HETATM 3725 C5 PLM A 415 −59.532 28.742 3.701 1.00 89.98 C HETATM 3726 C6 PLM A 415 −59.310 27.816 2.505 1.00 88.93 C HETATM 3727 C7 PLM A 415 −60.003 26.467 2.677 1.00 86.74 C HETATM 3728 C8 PLM A 415 −59.012 25.330 2.467 1.00 86.47 C HETATM 3729 C9 PLM A 415 −59.709 23.971 2.504 1.00 88.96 C HETATM 3730 CA PLM A 415 −58.837 22.893 3.156 1.00 90.43 C HETATM 3731 CB PLM A 415 −59.094 21.474 2.622 1.00 94.25 C HETATM 3732 CC PLM A 415 −57.863 20.775 2.016 1.00 96.09 C HETATM 3733 CD PLM A 415 −57.471 19.483 2.752 1.00 100.00 C HETATM 3734 CE PLM A 415 −56.635 18.544 1.872 1.00 101.35 C HETATM 3735 CF PLM A 415 −56.391 17.207 2.576 1.00 101.14 C HETATM 3736 CG PLM A 415 −55.695 16.199 1.664 1.00 99.15 C HETATM 3737 C35 12P A 416 −12.915 54.897 17.807 1.00 109.60 C HETATM 3738 O34 12P A 416 −13.949 54.683 18.777 1.00 110.80 O HETATM 3739 C33 12P A 416 −13.451 54.394 20.091 1.00 108.13 C HETATM 3740 C32 12P A 416 −13.568 55.622 20.982 1.00 105.37 C HETATM 3741 O31 12P A 416 −12.299 56.022 21.539 1.00 101.39 O HETATM 3742 C30 12P A 416 −12.283 57.340 22.133 1.00 102.00 C HETATM 3743 C29 12P A 416 −13.231 57.476 23.346 1.00 99.93 C HETATM 3744 O28 12P A 416 −14.580 57.575 22.874 1.00 94.22 O HETATM 3745 C27 12P A 416 −15.580 58.132 23.720 1.00 91.37 C HETATM 3746 C26 12P A 416 −16.528 58.912 22.820 1.00 89.49 C HETATM 3747 O25 12P A 416 −15.752 59.825 22.016 1.00 91.31 O HETATM 3748 C24 12P A 416 −16.081 59.833 20.621 1.00 89.90 C HETATM 3749 C23 12P A 416 −14.998 60.574 19.838 1.00 88.02 C HETATM 3750 O22 12P A 416 −14.838 61.904 20.352 1.00 86.45 O HETATM 3751 C21 12P A 416 −15.839 62.838 19.944 1.00 77.75 C HETATM 3752 C20 12P A 416 −15.146 63.916 19.149 1.00 82.56 C HETATM 3753 O19 12P A 416 −14.005 64.462 19.834 1.00 79.81 O HETATM 3754 C18 12P A 416 −14.323 65.263 20.988 1.00 86.00 C HETATM 3755 C17 12P A 416 −13.223 66.275 21.332 1.00 87.84 C HETATM 3756 O16 12P A 416 −11.998 65.967 20.653 1.00 93.31 O HETATM 3757 C15 12P A 416 −11.011 65.354 21.493 1.00 96.79 C HETATM 3758 O HOH A 501 −23.201 38.168 1.127 1.00 60.95 O HETATM 3759 O HOH A 502 −28.322 45.445 19.106 1.00 62.27 O HETATM 3760 O HOH A 503 −25.663 51.540 11.557 1.00 73.63 O HETATM 3761 O HOH A 504 −18.403 54.490 20.568 1.00 49.22 O HETATM 3762 O HOH A 505 −28.865 62.967 14.476 1.00 49.07 O HETATM 3763 O HOH A 506 −38.344 25.395 6.999 1.00 53.60 O HETATM 3764 O HOH A 507 −24.235 47.669 28.279 1.00 58.12 O HETATM 3765 O HOH A 508 −29.566 58.644 29.505 1.00 53.32 O HETATM 3766 O HOH A 509 −9.129 65.077 35.788 1.00 53.82 O HETATM 3767 O HOH A 510 −31.588 56.235 5.270 1.00 54.83 O HETATM 3768 O HOH A 511 −33.767 −0.936 2.970 1.00 76.89 O HETATM 3769 O HOH A 512 −36.831 52.163 25.686 1.00 59.37 O HETATM 3770 O HOH A 513 −20.293 51.784 5.369 1.00 57.17 O HETATM 3771 O HOH A 514 −33.710 48.038 30.053 1.00 55.19 O HETATM 3772 O HOH A 515 −27.498 71.737 31.097 1.00 72.77 O HETATM 3773 O HOH A 516 −38.419 75.003 17.409 1.00 63.18 O HETATM 3774 O HOH A 517 −13.008 52.291 33.720 1.00 54.29 O HETATM 3775 O HOH A 518 −25.417 49.449 2.526 1.00 84.88 O HETATM 3776 O HOH A 519 −44.492 24.103 10.594 1.00 70.04 O HETATM 3777 O HOH A 520 −53.192 34.656 −0.012 0.50 68.32 O HETATM 3778 O HOH A 521 −33.730 52.334 13.531 1.00 71.71 O HETATM 3779 O HOH A 522 −19.401 44.894 11.284 1.00 82.98 O HETATM 3780 O HOH A 523 −20.706 −4.455 5.029 1.00 74.00 O HETATM 3781 O HOH A 524 −18.878 44.843 24.435 1.00 71.79 O HETATM 3782 O HOH A 525 −26.266 47.546 12.074 1.00 96.88 O HETATM 3783 O HOH A 526 −35.818 54.612 25.448 1.00 65.23 O HETATM 3784 O HOH A 527 −27.911 63.928 17.098 1.00 56.73 O HETATM 3785 O HOH A 528 −34.136 24.300 12.374 1.00 64.85 O HETATM 3786 O HOH A 529 −31.485 13.403 16.427 1.00 55.45 O HETATM 3787 O HOH A 530 −33.229 46.525 27.509 1.00 62.72 O HETATM 3788 O HOH A 531 −37.000 56.927 26.470 1.00 69.85 O HETATM 3789 O HOH A 532 −38.272 23.691 9.991 1.00 74.37 O HETATM 3790 O HOH A 533 −23.738 45.700 30.214 1.00 70.87 O HETATM 3791 O HOH A 534 −35.547 18.917 9.368 1.00 68.76 O HETATM 3792 O HOH A 535 −27.520 46.018 35.709 1.00 73.73 O HETATM 3793 O HOH A 536 −11.169 52.639 27.107 1.00 53.63 O HETATM 3794 O HOH A 537 −35.161 36.603 6.920 1.00 84.70 O HETATM 3795 O HOH A 538 −13.331 64.631 16.826 1.00 78.41 O HETATM 3796 O HOH A 539 −15.737 37.989 13.501 1.00 74.20 O HETATM 3797 O HOH A 540 −17.612 49.885 19.741 1.00 67.69 O HETATM 3798 O HOH A 541 −28.871 56.041 4.930 1.00 62.51 O HETATM 3799 O HOH A 542 −18.100 62.470 17.192 1.00 64.11 O HETATM 3800 O HOH A 543 −39.876 5.502 10.338 1.00 78.73 O HETATM 3801 O HOH A 544 −39.732 −5.317 −4.531 1.00 75.67 O HETATM 3802 O HOH A 545 −19.865 59.621 17.222 1.00 67.78 O HETATM 3803 O HOH A 546 −37.397 27.672 6.120 1.00 59.62 O HETATM 3804 O HOH A 547 −19.199 49.833 44.410 1.00 55.49 O HETATM 3805 O HOH A 548 −35.618 21.645 8.827 1.00 61.27 O CONECT 597 1283 CONECT 1229 1277 CONECT 1277 1229 CONECT 1283 597 CONECT 2906 3635 CONECT 3535 3720 CONECT 3545 3546 3551 3555 CONECT 3546 3545 3547 3552 CONECT 3547 3546 3548 3553 CONECT 3548 3547 3549 3554 CONECT 3549 3548 3550 3555 CONECT 3550 3549 3556 CONECT 3551 3545 3560 CONECT 3552 3546 CONECT 3553 3547 CONECT 3554 3548 CONECT 3555 3545 3549 CONECT 3556 3550 CONECT 3557 3558 3563 3566 CONECT 3558 3557 3559 3564 CONECT 3559 3558 3560 3565 CONECT 3560 3551 3559 3561 CONECT 3561 3560 3562 3566 CONECT 3562 3561 3567 CONECT 3563 3557 CONECT 3564 3558 CONECT 3565 3559 CONECT 3566 3557 3561 CONECT 3567 3562 CONECT 3568 3569 3570 3571 3572 CONECT 3569 3568 CONECT 3570 3568 CONECT 3571 3568 CONECT 3572 3568 CONECT 3573 3574 3575 3576 3577 CONECT 3574 3573 CONECT 3575 3573 CONECT 3576 3573 CONECT 3577 3573 CONECT 3578 3579 3580 3581 3582 CONECT 3579 3578 CONECT 3580 3578 CONECT 3581 3578 CONECT 3582 3578 CONECT 3583 3584 3585 3586 3587 CONECT 3584 3583 CONECT 3585 3583 CONECT 3586 3583 CONECT 3587 3583 CONECT 3588 3589 3590 3591 3592 CONECT 3589 3588 CONECT 3590 3588 CONECT 3591 3588 CONECT 3592 3588 CONECT 3593 3594 3595 3596 3597 CONECT 3594 3593 CONECT 3595 3593 CONECT 3596 3593 CONECT 3597 3593 CONECT 3598 3599 CONECT 3599 3598 3600 3605 CONECT 3600 3599 3601 CONECT 3601 3600 3602 CONECT 3602 3601 3603 3604 CONECT 3603 3602 CONECT 3604 3602 CONECT 3605 3599 3606 CONECT 3606 3605 3607 CONECT 3607 3606 3608 3612 CONECT 3608 3607 3609 CONECT 3609 3608 3610 CONECT 3610 3609 3611 CONECT 3611 3610 3612 3613 CONECT 3612 3607 3611 3615 CONECT 3613 3611 3614 CONECT 3614 3613 3615 3616 CONECT 3615 3612 3614 3619 CONECT 3616 3614 3617 CONECT 3617 3616 3618 CONECT 3618 3617 3619 CONECT 3619 3615 3618 CONECT 3620 3621 3624 CONECT 3621 3620 3622 CONECT 3622 3621 3623 CONECT 3623 3622 3625 CONECT 3624 3620 CONECT 3625 3623 CONECT 3626 3627 3630 CONECT 3627 3626 3628 CONECT 3628 3627 3629 CONECT 3629 3628 3631 CONECT 3630 3626 CONECT 3631 3629 CONECT 3632 3633 3634 3635 CONECT 3633 3632 CONECT 3634 3632 CONECT 3635 2906 3632 CONECT 3636 3637 3645 CONECT 3637 3636 3638 CONECT 3638 3637 3639 3663 CONECT 3639 3638 3640 CONECT 3640 3639 3641 3645 CONECT 3641 3640 3642 CONECT 3642 3641 3643 CONECT 3643 3642 3644 3649 CONECT 3644 3643 3645 3646 CONECT 3645 3636 3640 3644 3654 CONECT 3646 3644 3647 CONECT 3647 3646 3648 CONECT 3648 3647 3649 3652 3653 CONECT 3649 3643 3648 3650 CONECT 3650 3649 3651 CONECT 3651 3650 3652 CONECT 3652 3648 3651 3655 CONECT 3653 3648 CONECT 3654 3645 CONECT 3655 3652 3656 3657 CONECT 3656 3655 CONECT 3657 3655 3658 CONECT 3658 3657 3659 CONECT 3659 3658 3660 CONECT 3660 3659 3661 3662 CONECT 3661 3660 CONECT 3662 3660 CONECT 3663 3638 CONECT 3664 3665 3673 CONECT 3665 3664 3666 CONECT 3666 3665 3667 3691 CONECT 3667 3666 3668 CONECT 3668 3667 3669 3673 CONECT 3669 3668 3670 CONECT 3670 3669 3671 CONECT 3671 3670 3672 3677 CONECT 3672 3671 3673 3674 CONECT 3673 3664 3668 3672 3682 CONECT 3674 3672 3675 CONECT 3675 3674 3676 CONECT 3676 3675 3677 3680 3681 CONECT 3677 3671 3676 3678 CONECT 3678 3677 3679 CONECT 3679 3678 3680 CONECT 3680 3676 3679 3683 CONECT 3681 3676 CONECT 3682 3673 CONECT 3683 3680 3684 3685 CONECT 3684 3683 CONECT 3685 3683 3686 CONECT 3686 3685 3687 CONECT 3687 3686 3688 CONECT 3688 3687 3689 3690 CONECT 3689 3688 CONECT 3690 3688 CONECT 3691 3666 CONECT 3692 3693 3701 CONECT 3693 3692 3694 CONECT 3694 3693 3695 3719 CONECT 3695 3694 3696 CONECT 3696 3695 3697 3701 CONECT 3697 3696 3698 CONECT 3698 3697 3699 CONECT 3699 3698 3700 3705 CONECT 3700 3699 3701 3702 CONECT 3701 3692 3696 3700 3710 CONECT 3702 3700 3703 CONECT 3703 3702 3704 CONECT 3704 3703 3705 3708 3709 CONECT 3705 3699 3704 3706 CONECT 3706 3705 3707 CONECT 3707 3706 3708 CONECT 3708 3704 3707 3711 CONECT 3709 3704 CONECT 3710 3701 CONECT 3711 3708 3712 3713 CONECT 3712 3711 CONECT 3713 3711 3714 CONECT 3714 3713 3715 CONECT 3715 3714 3716 CONECT 3716 3715 3717 3718 CONECT 3717 3716 CONECT 3718 3716 CONECT 3719 3694 CONECT 3720 3535 3721 3722 CONECT 3721 3720 CONECT 3722 3720 3723 CONECT 3723 3722 3724 CONECT 3724 3723 3725 CONECT 3725 3724 3726 CONECT 3726 3725 3727 CONECT 3727 3726 3728 CONECT 3728 3727 3729 CONECT 3729 3728 3730 CONECT 3730 3729 3731 CONECT 3731 3730 3732 CONECT 3732 3731 3733 CONECT 3733 3732 3734 CONECT 3734 3733 3735 CONECT 3735 3734 3736 CONECT 3736 3735 CONECT 3737 3738 CONECT 3738 3737 3739 CONECT 3739 3738 3740 CONECT 3740 3739 3741 CONECT 3741 3740 3742 CONECT 3742 3741 3743 CONECT 3743 3742 3744 CONECT 3744 3743 3745 CONECT 3745 3744 3746 CONECT 3746 3745 3747 CONECT 3747 3746 3748 CONECT 3748 3747 3749 CONECT 3749 3748 3750 CONECT 3750 3749 3751 CONECT 3751 3750 3752 CONECT 3752 3751 3753 CONECT 3753 3752 3754 CONECT 3754 3753 3755 CONECT 3755 3754 3756 CONECT 3756 3755 3757 CONECT 3757 3756 MASTER 455 0 16 19 4 0 18 6 3804 1 219 39 END 

1. A composition comprising a polypeptide in crystalline form, wherein said polypeptide comprises: a G-protein coupled receptor (GPCR) comprising an IC3 loop containing a substitution that comprises the amino acid sequence of a lysozyme.
 2. A composition comprising a fusion protein in crystalline form, wherein said fusion protein comprises, from N-terminus to C-terminus: a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4 and TM5 regions of said GPCR; b) a domain comprising the amino acid sequence of a lysozyme; c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR.
 3. The composition of claim 2, wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR.
 4. The composition of claim 1, wherein said GPCR is a β2-adrenergic receptor.
 5. The composition of claim 2, wherein said fusion protein is bound to a ligand for said GPCR.
 6. A composition comprising a polypeptide in crystalline form, wherein said polypeptide comprises, from N-terminus to C-terminus: a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the amino acid sequence that is N-terminal to the IC3 loop of said GPCR; b) a domain comprising the amino acid sequence of a lysozyme; c) a second portion of said GPCR, wherein said second portion comprises the amino acid sequence that is C-terminal to the IC3 loop of said GPCR.
 7. A crystalline fusion protein which comprises a beta-2 adrenergic receptor and lysozyme.
 8. The crystalline fusion protein of claim 7 wherein the lysozyme is a T4 lysozyme sequence.
 9. The crystalline fusion protein of claim 7 wherein the beta-2 adrenergic receptor has a E122W mutation.
 10. The crystalline fusion protein of claim 7 wherein the beta-2 adrenergic receptor has a E122W mutation and wherein the lysozyme is a T4 lysozyme sequence.
 11. The crystalline fusion protein of claim 7 wherein the lysozyme is T4 lysozyme sequence in place of the third intracellular loop of the beta-2 adrenergic receptor.
 12. The crystalline fusion protein of claim 7 wherein residues 231^(5.70) to 262^(6.24) are replaced by residues 2 to 161 of T4 lysozyme, and the beta-2 adrenergic receptor is truncated at position
 365. 13. The crystalline fusion protein of claim 7 further comprising a ligand non-covalently bound thereto.
 14. The crystalline fusion protein of claim 13 wherein the ligand is carazolol. 